KR20220107257A - Bispecific fusion proteins and their applications - Google Patents

Abstract

A bispecific fusion protein having a novel structure is provided. The second binding structural domain is inserted into the IgG hinge region in full length by any peptide joint. The fusion protein has the same expression and production advantages as IgG, and does not affect the binding activity of the Fab region of the fusion protein, thereby further improving stability and obtaining a higher half-life. In addition, the binding activity of the second binding structural domain to the target binding site was significantly improved compared to the binding activity of the monomer to the target corresponding to the soluble native binding fragment.

Description

Bispecific fusion proteins and their applications

The present invention relates to a bispecific fusion protein having a novel structure and applications thereof. The present invention also relates to polynucleotides, nucleic acid constructs and host cells encoding or making such fusion proteins.

With the development of biotechnology, antibodies are playing an important role in the treatment of tumors and immune diseases as mature therapeutic agents. Most antibody drugs on the market today target a single antigen and can act on only one target molecule. However, complex diseases in nature are often caused by multiple factors and are accompanied by upregulation of various receptors, including overexpression or synergistic effects of disease mediators, or molecular interactions of signaling networks. Therefore, modulating multiple signaling pathways may enhance the efficacy of therapeutic antibodies. Bispecific antibody (BsAb) therapy using a dual targeting strategy has become one of the effective treatments for tumors and immune diseases (Kontermann R. Dual targeting strategies with bispecific antibodies [C]//MAbs. Taylor & Francis, 2012, 4 (Kontermann R. Dual targeting strategies with bispecific antibodies [C]//MAbs. Taylor & Francis, 2012, 4) 2): 182-197.).

Bispecific antibodies can specifically bind to two different antigens or epitopes (Carter P. Bispecific human IgG by design [J]. Journal of immunological methods, 2001, 248(1-2): 7-15). In the 1980s, Morrison et al. prepared the first truly tetravalent bispecific antibodies against the dextran and dansyl groups by linking single-chain antibodies with different specificities using flexible peptides for fusion expression (Coloma M J, Morrison S L. Design and production of novel tetravalent bispecific antibodies [J]. Nature biotechnology, 1997, 15(2): 159.). However, the bottleneck of antibody manufacturing technology and lack of basic research on signaling pathways have hindered the development of bispecific antibodies. With a deep understanding of the pathogenesis of diseases and the rapid development of therapeutic monoclonal antibody (McAbs)-related technologies, the construction, expression and purification technology of antibodies has been greatly improved, and it is possible to overcome the limiting factors in the development of bispecific antibodies. It has the skills and motivation. Currently, the mechanisms of bispecific or multispecific antibodies are mainly as follows: (1) crosslinked cells (in trans); (2) receptor inhibition (in-cis) or receptor activation (in-cis); (3) cofactor mimetics; (4) PIgGyback approach (Aran F. Labrijn, et al. Bispecific antibodies: a mechanistic review of the pipeline, Nature Reviews Drug Discovery volume 18, pages 585-608 (2019)).

In the development of a bispecific antibody, the choice of molecular structure is very important. Each bispecific antibody design has advantages and disadvantages, but designing a bispecific antibody for clinical therapeutic purposes must address the following issues. First, the correct coupling or pairing of two pairs (or more) of different light and heavy chains must be ensured. Second, the independence of each binding domain of each monoclonal antibody is maintained, and steric interference should not occur when different epitopes are simultaneously bound. Third, the antibody molecule should be easy to express in mammalian cells without complex protein modification processes. Fourth, excellent drug properties such as high thermal stability, high chemical stability, high solubility, ease of polymerization, low viscosity, high expression, and appropriate half-life are required (Spiess C, Zhai Q, Carter P J. Alternative molecular formats) and therapeutic applications for bispecific antibodies [J] Molecular immunology, 2015, 67(2): 95-106.).

Depending on the different molecular structural forms (ie, components and methods of construction), bispecific antibodies can be divided into many types. For example, it is divided into a symmetric structure and an asymmetric structure according to the left-right symmetry of the structure, and can be divided into a bispecific antibody containing an Fc region and a bispecific antibody without an Fc region depending on the presence or absence of an Fc region, and antigen Depending on the number of binding domains, they are classified as bivalent, trivalent, tetravalent, or divided into polyvalent configurations. Currently, dozens of structures have been developed for bispecific antibodies (Spiess C, Zhai Q, Carter P J. Alternative molecule functions and therapy for bispecific antibody [J]. Molecular immunology, 2015, 67(2): 95-106.) , these bispecific antibodies have various structures and are divided into approximately five categories: IgG-like bispecific antibodies, IgG-like bispecific antibodies with additional functional regions, bispecific antibodies with different antigen-binding fragments linked in series, Fusion protein-type bispecific antibodies and chemically linked bispecific antibodies. Among them, the construction of IgG-like bispecific antibodies mainly uses recombinant DNA technology for structural modification. Since the prepared IgG-like bispecific antibody has a fully crystallizable fragment (ie, Fc fragment), Fc fragment-mediated ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-mediated cytotoxicity, complement-dependent cytotoxicity), etc. are maintained while maintaining It can bind other antigens. At the same time, this type of antibody also retains the properties of binding to the neonatal Fc receptor (FcRn) and extending the in vivo half-life of the antibody (Ridgway J B B, Presta L G, Carter P. 'Knobs-into-holes' engineering of antibody CH3). domains for heavy chain heterodimerization [J. Protein Engineering, Design and Selection, 1996, 9(7): 617-621.); IgG-like bispecific antibodies with additional functional regions are usually based on existing IgG antibodies and have additional functions such as specific antigen-binding fragments (i.e. single domain antibodies, single chain antibodies, etc.) that differ to the heavy and/or light chain via fusion proteins. region) (LaFleur D, Abramyan D, Kanakaraj P, et al. Monoclonal antibody therapeutics with up to five specificities: functional enhancement through fusion of target-specific peptides [C]//MAbs. Taylor & Francis, 2013 , 5(2): 208-218.); Bispecific antibodies in which different antigen-binding fragments are linked in series are obtained by linking different Fab, single chain antibodies, single domain antibodies or antigen-binding fragments of a specific sequence via a linking peptide (Stork R, Muller D, Kontermann R E. A novel tri-functional antibody fusion protein with improved pharmacokinetic properties generated by fusing a bispecific single-chain diabody with an albumin-binding domain from streptococcal protein G [J]. Protein Engineering, Design & Selection, 2007, 20(11): 569 -576.); A fusion protein-type bispecific antibody is a protein molecule capable of simultaneously binding to two or more antigens, and is formed by linking antibodies/antibody fragments (IgG, Fab, scFv, etc.) with different specificities through a linker such as a polypeptide fragment. ; And while chemically bound bispecific antibodies are similar to fusion protein-type bispecific antibodies, in the case of chemically bound bispecific antibodies, two antibodies/antibody fragments must be prepared separately first, and then these two antibodies undergo chemical bonding. or both antibodies are bound to the carrier protein.

In addition, with the development of basic research on signaling pathways and the expansion of the bispecific antibody mechanism, more and more targets and combinations for bispecific antibody selection are increasing, and PD-1, PD-L1, CTLA-4, LAG-3, FGL1 , TIM-3, galectin-9, TIGIT, CD155 and TGF-β receptor (TGF-βR), CD80, CD86, VEGFR, VEGF-trap, FGL1, CD70, 4-1BBL, OX40L and SIRPα; and Claudin 18.2, HER-2, Mesothelin, BCMA, SSTR2, GPRC5D, PSMA, FCRH5, CD33, CD123, CD20, A33, CEA, CD28, DLL3, EGFR, VEGFR, VEGFR2, VEGF-A, Nectin- 4, tumor antigens including FGFR, C-met, RANKL, PDGF, PDGFR, PDGFRα, DLL4, Ang-1 and Ang-2, and the like. Combining immune checkpoint targets or tumor targets with other targets involved in the developmental resistance of cancer cells or T-cell activation, or the combination of multiple immune checkpoint targets, greatly improves the therapeutic potential of malignant diseases such as tumors. In early stages, a combination of two separate checkpoint antibodies was used clinically to improve efficacy. For example, combined treatment of ipilimumab (anti-CTLA4) and nivolumab (anti-PD1) may improve survival in patients with melanoma compared to ipilimumab alone. Currently, the safety and early efficacy of four PD-1xCTLA4 bsAbs are being evaluated in early clinical trials. The concept of inhibitors that block two immune checkpoints is also used clinically for other target combinations such as PD-1 x LAG 3, PD-1 x TIM3 and PD-L1 x CTLA 4 (Wolchok, J. D. et al. Overall Survival). with Combined Nivolumab and Ipilimumab in Advanced Melanoma, N. Engl. J. Med. 377(14): 1345-1356, (2017); Dovedi, S. J. et al. MEDI5752: A novel bispecific antibody that preferentially targets CTLA-4 on PD -1 expressing T-cells. Cancer Res. 78 (13), Supplement. Abstract 2776: (2018). Hedvat, M. et al. Simultaneous checkpoint — checkpoint or checkpoint — costimulatory receptortargeting with bispecific antibodies promotes enhanced human T cell activation [ abstract P664].Presented at the 2018 Society for Immunotherapyof Cancer (SITC) (2018); Aran F. Labrijn, et al. Bispecific antibodies: a mechanistic review of the pipeline, Nature Reviews Drug Discovery volume 18, pages 585-608 (2019) ).

Currently, most of the bispecific antibodies are still in the clinical or preclinical research stage, and only three bispecific antibodies are currently licensed for the market. These are Trion Pharma's Catumaxomab (discontinued due to commercial factors in 2017), Amgen's Blinatumomab and Roche's Emicizumab. Catumaxomab is an IgG-like bispecific antibody in the form of a Triomab. It targets T cells and tumor cells overexpressing epithelial cell adhesion molecule (EpCAM) and inhibits tumor cells through T cell activation, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). It was approved by the EMA in 2009 for the treatment of malignant ascites due to EpCAM-positive tumors that are ineffective or ineffective with standard treatment, and is also the first bispecific antibody to receive marketing approval. Blinatumomab is a bispecific antibody lacking an Fc region, adopting the structural conformation of BiTE (Bispecific T Cell Engager). Blinatumomab induces T cell-mediated tumor cell killing effects by binding to T cells through the anti-CD3 moiety and to malignant and normal B cells through the anti-CD19 moiety. In 2014, blinatumomab received FDA approval for the treatment of Philadelphia chromosome-negative precursor B-cell acute lymphoblastic leukemia. Emicizumab is a modified humanized bispecific IgG4 monoclonal antibody capable of binding factor IXa and factor X. This antibody adopts "Knobs-into-Holes (KiH)" for Fc design, "Common Light Chain-IgG (CLC-IgG)" for LC design, and "Multidimensional Optimization" for variable region optimization, Factor VIII It received FDA approval in 2017 for routine prophylaxis of hemophilia A in the presence of an inhibitor.

Bispecific antibodies approved for marketing (including discontinuation of sale) bispecific antibody trade name research institute Year of initial approval katumaxomab Removab Trion Pharma 2009 (EMA)
(Delisted in 2017) blinatumomab Blincyto Amgen 2014 (FDA) emicizumab Hemlibra Roche 2017 (FDA)

ller D, Kontermann R E. A novel tri-functional antibody fusion protein with improved pharmacokinetic properties generated by fusing a bispecific single-chain diabody with an albumin-binding domain from streptococcal protein G [J]. Protein Engineering, Design & Selection, 2007, 20(11): 569-576.). 또한, 이러한 유형의 항체는 대부분 정제 과정에서 친화성 정제 수단(affinity purification means)을 채택하기 어렵기 때문에 정제 과정 및 완제품 내 불순물 분석도 기존의 IgG에 비해 복잡하며(Tan P H, Sandmaier B M, Stayton P S. Contributions of a highly conserved VH/VL hydrogen bonding interaction to scFv folding stability and refolding efficiency [J]. Biophysical journal, 1998, 75(3): 1473-1482.), 그 중 가장 대표적인 것이 블리나투모맙이다. 블리나투모맙은 Fc 단편이 없기 때문에 분자량이 작고, 혈액 내 반감기가 1.25±0.63h에 불과하여 온전한 항체의 반감기보다 훨씬 짧으므로, 유효량에 도달하기 위해서는 4주간의 지속적인 정맥 주사가 필요하며, 시약 사용 시에는 추가로 지속적인 주입 장치를 갖추어야 한다. 또한, 안정성이 좋지 않고 생산 중 응집체를 쉽게 형성하여 제품의 품질에 영향을 미치고 생산 비용을 증가시킨다. 현재 이중특이성 항체는 단일클론항체에 비해 낮은 발현, 낮은 안정성, 복잡한 생산공정, 현저히 높은 연구개발비 등 많은 문제점을 가지고 있어 이중특이성 항체의 개발을 제한하고 있다. 따라서 더 많은 임상 옵션을 제공할 수 있는 새로운 구조의 이중특이성 항체 개발이 시급하다.Although there are currently dozens of bispecific antibodies/platforms and R&D experience with three commercial drugs, the development of bispecific antibodies has more problems and challenges than general antibodies due to its complex structure and various functional properties. For example, IgG-like bispecific antibodies face the problem of heavy chain-heavy chain or heavy chain-light chain mismatch during manufacturing, the most representative of which is katumaxomab. Although katumaxomab is the first bispecific antibody to be approved for marketing, it has several very obvious limitations, mainly reflected in the complex production process of triomab antibodies and the immunogenicity issue of the relatively easy emergence of heterologous antibodies. Released but discontinued due to commercial factors; For another example, bispecific antibodies with different antigen-binding fragments linked in series have a short half-life due to the lack of a constant region, especially the Fc fragment, and the expression level and intracellular stability after expression are generally lower than that of conventional IgG. low (Stork R, M

ller D, Kontermann R E. A novel tri-functional antibody fusion protein with improved pharmacokinetic properties generated by fusing a bispecific single-chain diabody with an albumin-binding domain from streptococcal protein G [J]. Protein Engineering, Design & Selection, 2007, 20(11): 569-576.). In addition, most of these types of antibodies are difficult to adopt affinity purification means in the purification process, so the purification process and the analysis of impurities in finished products are more complicated than those of conventional IgG (Tan PH, Sandmaier BM, Stayton P). S. Contributions of a highly conserved VH/VL hydrogen bonding interaction to scFv folding stability and refolding efficiency [J]. Biophysical journal, 1998, 75(3): 1473-1482.), the most representative among them is blinatumomab . Since blinatumomab has no Fc fragment, its molecular weight is small, and its half-life in blood is only 1.25±0.63h, which is much shorter than the half-life of an intact antibody, so 4 weeks of continuous intravenous injection is required to reach an effective amount. In use, an additional continuous infusion device should be provided. In addition, it has poor stability and easily forms agglomerates during production, which affects the quality of the product and increases the production cost. Currently, bispecific antibodies have many problems such as low expression, low stability, complex production process, and significantly high R&D cost compared to monoclonal antibodies, limiting the development of bispecific antibodies. Therefore, there is an urgent need to develop a bispecific antibody with a novel structure that can provide more clinical options.

In order to solve the above problems, the present invention provides a bispecific fusion protein having a novel structure belonging to an IgG-like bispecific fusion protein having an additional functional region.

In order to solve the above problems, the present invention provides a bispecific fusion protein having a novel structure belonging to an IgG-like bispecific fusion protein having an additional functional region. Specifically, the fusion protein has a structure in which a second binding domain is inserted through an arbitrary peptide linker into the hinge region of full-length immunoglobulin G (IgG), and IgG The Fab region of is the first binding domain of the fusion protein; after inserting the second binding domain, the heavy chain of the IgG is the heavy chain of the fusion protein and the light chain of the IgG is the light chain of the fusion protein; the second binding domain is selected from a combination of a human receptor or ligand, an extracellular domain fragment of a receptor or ligand, a binding domain fragment, and a fragment of a receptor or ligand; wherein the receptor or ligand forms a dimerization or multimerization structure in a natural signaling pathway to activate or inhibit a signaling pathway, wherein the second binding domain and the first binding domain target different targets. The bispecific fusion protein having the above structure was named as a fusion protein having a Hibody (Hinge-insertion bispecific antibody) structure (hereinafter referred to as a hibody structure fusion protein or high body).

In addition, the first binding domain targets and binds an immune checkpoint molecule or a tumor antigen.

In addition, immune checkpoint molecules include PD-1 (programmed cell death protein 1), PD-L1 (programmed cell death 1 ligand 1), CTLA-4 (cytotoxic T lymphocyte-associated antigen-4), and LAG-3 (lymphocytes). activating gene-3), FGL1 (fibrinogen-like protein 1), TIM-3 (T cell immunoglobulin-3), galectin-9, TIGIT (T cell immunoreceptor with Ig and ITIM domains), CD155, CD47; Claudin 18.2, Her-2 (human epidermal growth factor receptor-2), Mesothelin, BCMA (B cell maturation antigen), SSTR2 (somatostatin receptor 2), GPRC5D (G-protein coupled receptor family C group 5 member D), PSMA ( Prostate specific membrane antigen), FcRH5 (Fc receptor-like protein 5), CD33, CD123, CD20, A33, CEA (carcinogenic antigen), CD28, DLL3 (delta-like protein 3), EGFR (epidermal growth factor receptor), VEGFR (vascular endothelial growth factor receptor), VEGFR2 (vascular endothelial growth factor receptor 2), VEGF-A (vascular endothelial growth factor-A), Nectin-4, FGFR (fibroblast growth factor receptor), C-met , RANKL (nuclear Receptor activator of factor kappa-B ligand), PDGF (platelet-derived growth factor), PDGFR (platelet-derived growth factor receptor), PDGFRα (platelet-derived growth factor receptor α), DLL4 (delta-like ligand 4), Ang-1 (anji tumor antigens including opoietin-1) and Ang-2 (angiopoietin-2).

In addition, the second binding domain is human TGF-β (modifying growth factor-β), CTLA-4, VEGF, LAG3, CD27, 4-1BB (CD137), OX40 (CD134), CD47, FGL1 (fibrinogen-like protein 1) , TLT-2 (Trem-like transcript 2), CD28, HGF (hepatocyte growth factor), CSF1 (colony stimulating factor 1), CXCL1 (CXC chemokine ligand 1), CXCL2 (CXC chemokine ligand 2), CXCL3 (CXC chemokine ligand) Ligand 3), CXCL5 (CXC chemokine ligand 5), CXCL6 (CXC chemokine ligand 6), CXCL7 (CXC chemokine ligand 7), CXCL8 (CXC chemokine ligand 8), CXCL9 (CXC chemokine ligand 9), CXCL10 (CXC chemokine ligand 10) ), CXCCL12 (CXC chemokine ligand 12), GITR (glucocorticoid-induced tumor necrosis factor receptor), EGF (epidermal growth factor) and ICOSL (inducible co-stimulator ligand).

In addition, the receptor or ligand is human TGF-β receptor (TGF-βR), CD80, CD86, VEGFR, VEGF-trap, FGL1, CD70, 4-1BBL, OX40L, SIRPα (signal regulatory protein α), B7-H3 ( CD276), C-met, CSF1R (colony stimulating factor 1 receptor), CXCR2 (CXC chemokine receptor 2), CXCR3 (CXC chemokine receptor 3), CXCR4 (CXC chemokine receptor 4), GITRL (glucocorticoid-induced TNF receptor ligand), EGFR and ICOS (inducible co-stimulants).

Among them, CD80 and CD86 are transmembrane glycoproteins that are members of the immunoglobulin superfamily (IgSF). The mature CD80 molecule consists of 254 amino acids, including 208 amino acids in the extracellular domain (ECD), 25 amino acids in the transmembrane domain, and 21 amino acids in the intracellular domain. Similarly, a mature CD86 molecule consists of 303 amino acids, including 222 amino acids in the extracellular domain, 20 amino acids in the transmembrane domain, and 61 amino acids in the intracellular domain. The extracellular domains of CD80 and CD86 include immunoglobulin V (IgV) and immunoglobulin C (IgC) regions. CD80 and CD86 bind to the ligands CD28 and CTLA-4 through the immunoglobulin V (IgV) region. For CD80 and CD86 binding to CD28, CD80 and CD86 produce antigen-induced T cell activation, proliferation and regulator function. CD80 and CD86 act as negative regulators by down-regulating the immune response when CD80 and CD86 bind to CTLA-4. Therefore, CD80 and CD86 play important roles in autoimmune monitoring, humoral immune response, and transplantation response as costimulatory factors when T lymphocytes are activated. In contrast to activating T cells using an agonistic anti-CD28 antibody, activating T cells using the ligand CD80 of CD28 as an activator does not cause a severe cytokine storm, potentially putting the patient's life at risk. greatly reduced

In some embodiments, the CD80 ECD is selected from human CD80 (eg, human CD80 of SEQ ID NO: 134) or human CD80 ECD derived from CD80 isoform 2 or isoform 3 (SEQ ID NOs: 135 and 136). The CD80 ECD comprises a CD80 immunoglobulin V (IgV) region (CD80-IgV, SEQ ID NO: 133). In one embodiment, the CD80 ECD comprises a human CD80 immunoglobulin V region and a C region (CD80-IgVIgC, SEQ ID NO:32). In one embodiment, the CD80 ECD is a human CD80 ECD. In one embodiment, the CD80 ECD comprises human CD80 IgV.

CD80-IgV (SEQ ID NO: 133):

VIHVTKEVKE VATLSCGHNV SVEELAQTRI YWQKEKKMVL TMMSGDMNIW PEYKNRTIFD 60

ITNNLSIVIL ALRPSDEGTY ECVVLKYEKD AFKREHLAEV TLSVKADFPT PS 112

CD80-IgVIgC (SEQ ID NO: 32):

VIHVTKEVKE VATLSCGHNV SVEELAQTRI YWQKEKKMVL TMMSGDMNIW PEYKNRTIFD 60

ITNNLSIVIL ALRPSDEGTY ECVVLKYEKD AFKREHLAEV TLSVKADFPT PSISDFEIPT 120

SNIRRIICST SGGFPEPHLS WLENGEELNA INTTVSQDPE TELYAVSSKL DFNMTTNHSF 180

MCLIKYGHLR VNQTFNWN 198

CD80 isotype 1 (SEQ ID NO: 134):

MGHTRRQGTS PSKCPYLNFF QLLVLAGLSH FCSGVIHVTK EVKEVATLSC GHNVSVEELA 60

QTRIYWQKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK 120

YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE 180

ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP 240

DNLLPSWAIT LISVNGFVI CCLTYCFAPR CRRRRNERL RRESVRPV 288

CD80 isotype 2 (SEQ ID NO: 135):

MGHTRRQGTS PSKCPYLNFF QLLVLAGLSH FCSGVIHVTK EVKEVATLSC GHNVSVEELA 60

QTRIYWQKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK 120

YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE 180

ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTSFAPRCR 240

ERRRNERLRR ESVRPV 256

CD80 isotype 3 (SEQ ID NO: 136):

MGHTRRQGTS PSKCPYLNFF QLLVLAGLSH FCSGVIHVTK EVKEVATLSC GHNVSVEELA 60

QTRIYWQKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK 120

YEKDAFKREH LAEVTLSVKG FAPRCRERRR NERLRRESVR PV 162

Further, the first binding domain and the second binding domain are selected from the following combinations:

(1) the first binding domain targets and binds to PD-L1, and the second binding domain is human TGF-β, VEGF, FGL1, CD47, CD155, HGF, CSF1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, target and bind to CXCL7, CXCL8, CXCL9, CXCL10, CXCL12, EGF or ICOSL; or

(2) the first binding domain targets and binds to PD-1, and the second binding domain targets and binds human CTLA-4, VEGF, HGF, EGF, CD28, LAG3, CD27, 4-1BB or OX40 do or; or

(3) the first binding domain targets and binds CTLA-4 and the second binding domain targets and binds human CD28, VEGF, HGF, EGF, LAG3, CD27, 4-1BB or OX40; or

(4) the first binding domain targets and binds LAG-3 and the second binding domain targets and binds human CD28, VEGF, HGF, EGF, CTLA-4, CD27, 4-1BB or OX40; or

(5) the first binding domain targets and binds FGL1, and the second binding domain is human TGF-β, VEGF, CD47, CD155, HGF, CSF1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, target and bind CXCL9, CXCL10, CXCL12, EGF or ICOSL; or

(6) the first binding domain targets and binds TIM-3 and the second binding domain targets and binds human VEGF, HGF, EGF, LAG3, CD27, 4-1BB or OX40; or

(7) the first binding domain targets and binds to galectin-9, and the second binding domain is human TGF-β, VEGF, CD47, CD155, HGF, CSF1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7 , binds by targeting CXCL8, CXCL9, CXCL10, CXCL12, EGF or ICOSL; or

(8) the first binding domain targets and binds TIGIT and the second binding domain targets and binds human TGF-β, HGF, EGF or VEGF; or

(9) the first binding domain targets and binds CD155, and the second binding domain targets and binds human TGF-β, VEGF, HGF, EGF, CD47 or CD155; or

(10) the first binding domain is claudin 18.2, HER-2, mesothelin, BCMA, SSTR2, GPRC5D, PSMA, FCRH5, CD33, CD123, CD20, A33, CEA, CD28, DLL3, EGFR, VEGFR, VEGFR2, VEGF- Targets and binds A, Nectin-4, FGFR, C-met, RANKL, PDGF, PDGFR, PDGFRα, DLL-4, Ang-1 or Ang-2, wherein the second binding domain is human CTLA-4, TGF- Binding by targeting β, VEGF, FGL1, LAG3, 4-1BB, OX40, CD27, CD28, CD47, CD155, HGF, CSF1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10 or ICOSL do.

In addition, the first binding domain and the receptor or ligand are selected from the following combinations:

(1) the first binding domain binds by targeting PD-L1, and the receptor or ligand is TGF-βRII, VEGFR, LAG-3, SIRPα, TIGIT, C-MET, CSF1R, CXCR2, CXCR3, CXCR4, EGFR or selected from ICOS; or

(2) the first binding domain targets and binds PD-1 and the receptor or ligand is selected from human CD80, CD86, VEGFR, c-MET, EGFR, FGL1, CD70, 4-1BBL or OX40L; or

(3) the first binding domain targets and binds CTLA-4, and the receptor or ligand is selected from human CD80, CD86, VEGFR, c-MET, EGFR, FGL1, CD70, 4-1BBL or OX40L; or

(4) the first binding domain targets and binds LAG-3, and the receptor or ligand is selected from human VEGFR, c-MET, EGFR, CD80, CD86, CD70, 4-1BBL or OX40L; or

(5) the first binding domain binds by targeting FGL1, and the receptor or ligand is selected from human TGF-βRII, VEGFR, SIRPα, TIGIT, c-MET, CSF1R, CXCR2, CXCR3, CXCR4, EGFR or ICOS; or

(6) the first binding domain targets and binds TIM-3, and the receptor or ligand is selected from human VEGFR, c-MET, EGFR, FGL1, CD70, 4-1BBL or OX40L; or

(7) the first binding domain targets and binds galectin-9, and the receptor or ligand is from human TGF-βRII, VEGFR, SIRPα, TIGIT, c-MET, CSF1R, CXCR2, CXCR3, CXCR4, EGFR or ICOS selected; or

(8) the first binding domain targets and binds TIGIT, and the receptor or ligand is selected from human TGF-βRII, c-MET, EGFR or VEGFR; or

(9) the first binding domain targets and binds CD155, and the receptor or ligand is selected from human TGF-βRII, VEGFR, c-MET, EGFR, SIRPα or TIGIT; or

(10) the first binding domain is claudin 18.2, HER-2, mesothelin, BCMA, SSTR2, GPRC5D, PSMA, FCRH5, CD33, CD123, CD20, A33, CEA, CD28, DLL3, EGFR, VEGFR, VEGFR2, Nectin- 4, FGFR, c-MET, RANKL, PDGF, PDGFR, PDGFRα, DLL4, Ang-1 or Ang-2 by targeting and binding, the receptor or ligand is human CTLA-4, CD80, CD86, TGF-βRII, VEGFR , FGL1, LAG3, 4-1BB, OX40, CD27, CD28, CD70, c-MET, SIRPα, TIGIT, CSF1R, CXCR2, CXCR3, CXCR4, EGFR or ICOS.

In addition, the fragment of the receptor or ligand includes a fragment of an extracellular domain and a fragment of the binding domain of the receptor or ligand.

In addition, the first binding domain targets and binds PD-L1; the second binding domain is a fragment of human TGF-βRII; Or the first binding domain binds to the target PD-1, the second binding domain is human CD80 ECD, CD80 IgV region, human CD80 IgVIgC, VEGFR1 ECD, VEGFR2 ECD or a second extracellular region of VEGFR1 and VEGFR2 3 comprising a combination of extracellular regions; or the first binding domain binds to and targets claudin 18.2, HER-2 or EGFR, and the second binding domain is a human CD80 ECD, a CD80 IgV region, a human CD80 IgVIgC, a VEGFR1 ECD, a VEGFR2 ECD or a second extracellular of VEGFR1 region and a third extracellular region of VEGFR2.

Further, the second binding domain is selected from:

(1) 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 compared to the sequence shown in SEQ ID NO: 31, 131 or 132, or the sequence shown in SEQ ID NO: 31, 131 or 132 % homology, or an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions or deletions on the sequence shown in SEQ ID NO: 31, 131 or 132 , human TGF-βRII; or

(2) at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology to the sequence shown in SEQ ID NO: 32 or 133, or to the sequence shown in SEQ ID NO: 32 or 133 a CD80 ECD comprising an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions or deletions in the sequence shown in SEQ ID NO: 32 or 133; or

(3) a combination of the second extracellular region of VEGFR1 and the third extracellular region of VEGFR2, wherein the sequence shown in SEQ ID NO: 33, or 80%, 85%, 90%, 95% compared to the sequence shown in SEQ ID NO: 33 , 96%, 97%, 98%, 99% or more homology, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions on the sequence shown in SEQ ID NO: 33 or fruit.

In addition, there is a peptide linker at the C-terminus and/or N-terminus of the second binding domain, and the peptide linker consists of 2-30 amino acids.

Additionally, the peptide linker may be:

(1) (GGGGS)n; or

(2) AKTTPKLEEEGEFSEAR (SEQ ID NO: 80); or

(3) AKTTPKLEEEGEFSEARV (SEQ ID NO: 81); or

(4) AKTTPKLGG (SEQ ID NO: 82); or

(5) SAKTTPKLGG (SEQ ID NO: 83); or

(6) SAKTTP (SEQ ID NO: 84); or

(7) RADAAP (SEQ ID NO: 85); or

(8) RADAAPTVS (SEQ ID NO: 86); or

(9) RADAAAAGGPGS (SEQ ID NO: 87); or

(10) RADAAAA (SEQ ID NO: 88); or

(11) SAKTTPKLEEEGEFSEARV (SEQ ID NO: 89); or

(12) ADAAP (SEQ ID NO: 90); or

(13) DAAPTVSIFPP (SEQ ID NO: 91); or

(14) TVAAP (SEQ ID NO: 92); or

(15) TVAAPSVFIFFP (SEQ ID NO: 93); or

(16) QPKAAP (SEQ ID NO: 94); or

(17) QPKAAPSVTLFPP (SEQ ID NO: 95); or

(18) AKTTPP (SEQ ID NO: 96); or

(19) AKTTPPSVTPLAP (SEQ ID NO: 97); or

(20) AKTTAP (SEQ ID NO: 98); or

(21) AKTTAPSVYPLAP (SEQ ID NO: 99); or

(22) ASTKGP (SEQ ID NO: 100); or

(23) ASTKGPSVFPLAP (SEQ ID NO: 101); or

(24) GENKVEYAPALMALS (SEQ ID NO: 102); or

(25) GPAKELTPLKEAKVS (SEQ ID NO: 103); or

(26) GHEAAAVMQVQYPAS (SEQ ID NO: 104); or

(27) GGGGSGGGGSGGGGSA (SEQ ID NO: 105),

wherein n is 1, 2, 3 or 4;

when n=1, (GGGGS)n is GGGGS (SEQ ID NO: 120);

when n=2, (GGGGS)n is GGGGSGGGGS (SEQ ID NO: 121) or (GGGGS) ₂ ;

when n=3, (GGGGS)n is GGGGSGGGGSGGGGS (SEQ ID NO: 122) or (GGGGS) ₃ ;

When n=4, (GGGGS)n is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 123) or (GGGGS) ₄ .

Further, the size of the inserted second binding domain does not exceed 235, 240, 250, 260, 270, 280, 290 or 300 amino acids.

In addition, the insertion site of the second binding domain is located in the anterior central portion of the hinge region, the insertion site does not affect the formation of disulfide bonds in immunoglobulins, and the anterior central portion of the hinge region is generally the portion before 231A indicates. In addition, some amino acids in the hinge region before and after the insertion site are substituted or deleted. For example, the hinge region comprises a D221G and/or C220V mutation.

Also, the IgG is selected from mammalian IgG, humanized IgG and human IgG, mammals including mouse, rat and rabbit; Preferably, the IgG is IgG1, IgG2, IgG3 or IgG4.

In addition, the Fc region of the fusion protein is aglycosylated or deglycosylated, reduced fucosylation, or afucosylated.

Also, the IgG is atezolizumab, avelumab, durvalumab, nivolumab, pembrolizumab, semiflimab or ipilimumab. Preferably, the IgG is atezolizumab, the amino acid sequence of the heavy chain of the IgG is shown in SEQ ID NO: 106 and the amino acid sequence of the light chain of the IgG is shown in SEQ ID NO: 107; or the IgG is avelumab, the amino acid sequence of the heavy chain of IgG is set forth in SEQ ID NO: 108 and the amino acid sequence of the light chain of IgG is set forth in SEQ ID NO: 109; or the IgG is durvalumab, the amino acid sequence of the heavy chain of IgG is set forth in SEQ ID NO: 110 and the amino acid sequence of the light chain of IgG is set forth in SEQ ID NO: 111; or the IgG is nivolumab, the amino acid sequence of the heavy chain of IgG is set forth in SEQ ID NO: 112 and the amino acid sequence of the light chain of IgG is set forth in SEQ ID NO: 113; or the IgG is pembrolizumab, the amino acid sequence of the heavy chain of IgG is set forth in SEQ ID NO: 114 and the amino acid sequence of the light chain of IgG is set forth in SEQ ID NO: 115; or the IgG is ipilimumab, the amino acid sequence of the heavy chain of IgG is set forth in SEQ ID NO: 116 and the amino acid sequence of the light chain of IgG is set forth in SEQ ID NO: 117; or the IgG is semipliumab, the amino acid sequence of the heavy chain of IgG is set forth in SEQ ID NO: 118 and the amino acid sequence of the light chain of IgG is set forth in SEQ ID NO: 119.

Specifically, the present invention provides a high body structural fusion protein having a first binding domain targeting human PD-L1, wherein the CDRs of the heavy chain variable region and/or the CDRs of the light chain variable region in the Fab of IgG are defined by the following sequences has the same CDR sequence as the antibody, or has 1-2 amino acid substitutions in the CDR of the antibody as defined by the following sequence, wherein the antibody is defined as follows:

(1) the amino acid sequence of the heavy chain variable region of SEQ ID NO: 66; and/or

(2) the amino acid sequence of the light chain variable region of SEQ ID NO: 67.

In addition, the CDRs of the heavy chain variable region and/or the CDRs of the light chain variable region in the Fab of IgG are as follows:

(1) for the heavy chain variable region, the amino acid sequence of CDR1 is selected from amino acid sequences having 1 or 2 amino acid substitutions in SEQ ID NO: 1-5 and SEQ ID NO: 1-5; the amino acid sequence of CDR2 is selected from amino acid sequences having 1 or 2 amino acid substitutions in SEQ ID NO: 6-10 and SEQ ID NO: 6-10; the amino acid sequence of CDR3 is selected from the amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 11-15 and SEQ ID NO: 11-15; and/or

(2) for the light chain variable region, the amino acid sequence of CDR1 is selected from amino acid sequences having 1 or 2 amino acid substitutions in SEQ ID NOs: 16-20 and SEQ ID NOs: 16-20; the amino acid sequence of CDR2 is selected from the amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 21-25 and SEQ ID NO: 21-25; The amino acid sequence of CDR3 is selected from amino acid sequences having 1 or 2 amino acid substitutions in SEQ ID NOs: 26-30 and SEQ ID NOs: 26-30.

In certain embodiments, the complementarity determining regions CDRs 1-3 of the corresponding heavy and light chain variable regions, depending on the different measurement methods or system identification used, are set forth in Table 2.

Amino acid sequences of heavy and light chain variable region CDRs 1-3 of anti-PD-L1 antibody category system CDR1 CDR2 CDR3 heavy chain Chothia SEQ ID NO: 1
GFSLSRY SEQ ID NO: 6
WGVGT SEQ ID NO: 11
NWGTADYFDY AbM SEQ ID NO: 2
GFSLSRYSVH SEQ ID NO: 7
MIWGVGTTD SEQ ID NO: 12
NWGTADYFDY Kabat SEQ ID NO: 3RYSVH SEQ ID NO: 8
MIWGVGTTDYNSALKS SEQ ID NO: 13
NWGTADYFDY Contact SEQ ID NO: 4SRYSVH SEQ ID NO: 9
WLGMIWGVGTTD SEQ ID NO: 14
ARNWGTADYFD IMGT SEQ ID NO: 5GFSLSRYS SEQ ID NO: 10
IWGVGTT SEQ ID NO: 15
ARNWGTADYFDY light chain Chothia SEQ ID NO: 16
RASKSVHTSGYSYMH SEQ ID NO: 21
LASNLES SEQ ID NO: 26
QHSGELPYT AbM SEQ ID NO: 17
RASKSVHTSGYSYMH SEQ ID NO: 22
LASNLES SEQ ID NO: 27
QHSGELPYT Kabat SEQ ID NO: 18RASKSVHTSGYSYMH SEQ ID NO: 23
LASNLES SEQ ID NO: 28
QHSGELPYT Contact SEQ ID NO: 19HTSGYSYMHWY SEQ ID NO: 24
LLIYLASNLE SEQ ID NO: 29
QHSGELPY IMGT SEQ ID NO: 20KSVHTSGYSY SEQ ID NO: 25
LAS SEQ ID NO: 30
QHSGELPYT

In addition, the fusion protein comprises the following CDR sequences,

(1) the amino acid sequence of CDRs 1-3 of the heavy chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 1, 6, 11, or SEQ ID NO: 1, 6, 11; and/or the amino acid sequence of CDRs 1-3 of the light chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 16, 21, 26, or SEQ ID NO: 16, 21, 26; or

(2) the amino acid sequence of CDRs 1-3 of the heavy chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 2, 7, 12, or SEQ ID NO: 2, 7, 12; and/or the amino acid sequence of CDRs 1-3 of the light chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 17, 22, 27, or SEQ ID NO: 17, 22, 27; or

(3) the amino acid sequence of CDRs 1-3 of the heavy chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 3, 8, 13 or SEQ ID NO: 3, 8, 13; and/or the amino acid sequence of CDRs 1-3 of the light chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 18, 23, 28, or SEQ ID NO: 18, 23, 28; or

(4) the amino acid sequence of CDRs 1-3 of the heavy chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 4, 9, 14, or SEQ ID NO: 4, 9, 14; and/or the amino acid sequence of CDRs 1-3 of the light chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 19, 24, 29, or SEQ ID NO: 19, 24, 29; or

(5) the amino acid sequence of CDRs 1-3 of the heavy chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 5, 10, 15 or SEQ ID NO: 5, 10, 15; and/or the amino acid sequence of CDRs 1-3 of the light chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 20, 25, 30, or SEQ ID NO: 20, 25, 30.

In addition, the amino acid sequences of CDRs 1-3 of the heavy chain variable region in the heavy chain of the fusion protein are SEQ ID NOs: 3, 8, and 13; and/or the amino acid sequences of CDRs 1-3 of the light chain variable region are SEQ ID NOs: 18, 23, 28.

In addition, the sequences of the heavy chain variable region and the light chain variable region of the fusion protein are as follows.

(1) the heavy chain variable region has the sequence shown in SEQ ID NO: 66, or CDRs 1-3 identical to SEQ ID NO: 66, and has 80%, 85%, 90%, 95%, 96 homology compared to SEQ ID NO: 66 %, 97%, 98%, 99% or more of the sequence; and/or

(2) the light chain variable region has the sequence shown in SEQ ID NO: 67, or CDRs 1-3 identical to that of SEQ ID NO: 67, and has 80%, 85%, 90%, 95%, 96% homology compared to SEQ ID NO: 67 , 97%, 98%, 99% or more of the sequence.

More specifically, the amino acid sequences of the heavy and light chains of the fusion protein are as follows:

(1) the heavy chain of the fusion protein has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence shown in SEQ ID NO: 72, or SEQ ID NO: 72 have a sequence with;

(2) the light chain of the fusion protein has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology compared to the amino acid sequence shown in SEQ ID NO: 73, or SEQ ID NO: 73 have a sequence

In addition, the amino acid sequences of the heavy and light chains of the fusion protein are as follows.

(1) the amino acid sequence of the heavy chain of the fusion protein has 1-15 amino acid site mutations compared to SEQ ID NO: 72 or has an alternative peptide linker;

(2) the amino acid sequence of the light chain of the fusion protein has 1-10 amino acid site mutations compared to SEQ ID NO: 73.

Specifically, the present invention provides a high body structural fusion protein having a first binding domain targeting human PD-1, wherein the CDRs of the heavy chain variable region and/or the CDRs of the light chain variable region in the Fab are defined by the sequences has the same CDR sequence as the antibody, or 1-2 amino acid substitutions in the CDR of the antibody as defined by the following sequence, wherein the antibody is defined as follows:

(1) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 64; and/or

(2) The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 65.

In addition, the CDRs of the heavy chain variable region and/or the light chain variable region of the Fab in the fusion protein are as follows:

(1) for the heavy chain variable region, the amino acid sequence of CDR1 is selected from amino acid sequences having 1 or 2 amino acid substitutions in SEQ ID NOs: 34-38 and SEQ ID NOs: 34-38; the amino acid sequence of CDR2 is selected from amino acid sequences having 1 or 2 amino acid substitutions in SEQ ID NOs: 39-43 and SEQ ID NOs: 39-43; the amino acid sequence of CDR3 is selected from amino acid sequences having 1 or 2 amino acid substitutions in SEQ ID NOs: 44-48 and SEQ ID NOs: 44-48; and/or

(2) for the light chain variable region, the amino acid sequence of CDR1 is selected from the amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NOs: 49-53 and SEQ ID NOs: 49-53; the amino acid sequence of CDR2 is selected from an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NOs: 54-58 and SEQ ID NOs: 54-58; The amino acid sequence of CDR3 is selected from amino acid sequences having 1 or 2 amino acid substitutions in SEQ ID NOs: 59-63 and SEQ ID NOs: 59-63.

In certain embodiments, the complementarity determining regions CDRs 1-3 of the corresponding heavy and light chain variable regions, depending on the different measurement methods or system identification used, are set forth in Table 3.

Amino acid sequences of heavy and light chain variable region CDRs 1-3 of anti-PD-1 antibody category system CDR1 CDR2 CDR3 heavy chain Chothia SEQ ID NO: 34
GITFSNS SEQ ID NO: 39
WYDGSK SEQ ID NO: 44
NDDY AbM SEQ ID NO: 35
GITFSNSGMH SEQ ID NO: 40
VIWYDGSKRY SEQ ID NO: 45
NDDY Kabat SEQ ID NO: 36NSGMH SEQ ID NO: 41
VIWYDGSKRYYADSVKG SEQ ID NO: 46
NDDY Contact SEQ ID NO: 37SNSGMH SEQ ID NO: 42
WVAVIWYDGSKRY SEQ ID NO: 47
ATNDD IMGT SEQ ID NO: 38GITFSNSG SEQ ID NO: 43
IWYDGSKR SEQ ID NO: 48
ATNDDY light chain Chothia SEQ ID NO: 49
RASQSVSSYLA SEQ ID NO: 54
DASNRAT SEQ ID NO: 59
QQSSNWPRT AbM SEQ ID NO: 50
RASQSVSSYLA SEQ ID NO: 55
DASNRAT SEQ ID NO: 60
QQSSNWPRT Kabat SEQ ID NO: 51RASQSVSSYLA SEQ ID NO: 56
DASNRAT SEQ ID NO: 61
QQSSNWPRT Contact SEQ ID NO: 52SSYLAWY SEQ ID NO: 57
LLIYDASNRA SEQ ID NO: 62
QQSSNWPR IMGT SEQ ID NO: 53QSVSSY SEQ ID NO: 58
DAS SEQ ID NO: 63
QQSSNWPRT

In addition, CDRs 1-3 of the variable region of the fusion protein are defined as follows:

(1) the amino acid sequence of CDRs 1-3 of the heavy chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 34, 39, 44 or SEQ ID NO: 34, 39, 44; and/or the amino acid sequence of CDRs 1-3 of the light chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 49, 54, 59, or SEQ ID NO: 49, 54, 59; or

(2) the amino acid sequence of CDRs 1-3 of the heavy chain variable region is an amino acid sequence having one or two amino acid substitutions in SEQ ID NO: 35, 40, 45, or SEQ ID NO: 35, 40, 45; and/or the amino acid sequence of CDRs 1-3 of the light chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 50, 55, 60, or SEQ ID NO: 50, 55, 60; or

(3) the amino acid sequence of CDRs 1-3 of the heavy chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 36, 41, 46 or SEQ ID NO: 36, 41, 46; and/or the amino acid sequence of CDRs 1-3 of the light chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 51, 56, 61, or SEQ ID NO: 51, 56, 61; or

(4) the amino acid sequence of CDRs 1-3 of the heavy chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 37, 42, 47, or SEQ ID NO: 37, 42, 47; and/or the amino acid sequence of CDRs 1-3 of the light chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 52, 57, 62, or SEQ ID NO: 52, 57, 62; or

(5) the amino acid sequence of CDRs 1-3 of the heavy chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 38, 43, 48 or SEQ ID NO: 38, 43, 48; and/or the amino acid sequence of CDRs 1-3 of the light chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 53, 58, 63, or SEQ ID NO: 53, 58, 63.

Further, CDRs 1-3 of the variable region of the fusion protein are defined as follows: the amino acid sequences of CDRs 1-3 of the heavy chain variable region are SEQ ID NOs: 36, 41, 46; and/or the amino acid sequences of CDRs 1-3 of the light chain variable region are SEQ ID NOs: 51, 56, 61.

In addition, the amino acid sequences of the heavy chain variable region and the light chain variable region of the fusion protein are as follows.

(1) the heavy chain variable region has the sequence shown in SEQ ID NO: 64, or CDRs 1-3 identical to that of SEQ ID NO: 64, and has 80%, 85%, 90%, 95%, 96% homology compared to SEQ ID NO: 64 , 97%, 98%, 99% or more of the sequence; and/or

(2) the light chain variable region has the sequence shown in SEQ ID NO: 65, or CDRs 1-3 identical to SEQ ID NO: 65, and has 80%, 85%, 90%, 95%, 96% homology compared to SEQ ID NO: 65; 97%, 98%, 99% or more sequences.

More specifically, the amino acid sequences of the heavy and light chains of the fusion protein are defined as follows:

(1) the heavy chain of the fusion protein has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence shown in SEQ ID NO: 68, or SEQ ID NO: 68 have a sequence with;

(2) the light chain of the fusion protein has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology compared to the amino acid sequence shown in SEQ ID NO: 69, or SEQ ID NO: 69 have a sequence

In addition, the amino acid sequences of the heavy and light chains of the fusion protein are defined as follows:

(1) the amino acid sequence of the heavy chain of the fusion protein has 1-15 amino acid site mutations compared to SEQ ID NO: 68 or has an alternative peptide linker;

(2) the amino acid sequence of the light chain of the fusion protein has 1-10 amino acid site mutations compared to SEQ ID NO: 69.

More specifically, the amino acid sequences of the heavy and light chains of the fusion protein are defined as follows:

(1) the heavy chain of the fusion protein has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence shown in SEQ ID NO: 70, or SEQ ID NO: 70 have a sequence with;

(2) the light chain of the fusion protein has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence shown in SEQ ID NO: 71, or SEQ ID NO: 71 have a sequence with

In addition, the amino acid sequences of the heavy and light chains of the fusion protein are defined as follows:

(1) the amino acid sequence of the heavy chain of the fusion protein has 1-15 amino acid site mutations compared to SEQ ID NO: 70 or has an alternative peptide linker;

(2) the amino acid sequence of the light chain of the fusion protein has 1-10 amino acid site mutations compared to SEQ ID NO: 71.

The present invention also provides an isolated polynucleotide encoding the fusion protein.

The present invention also provides a nucleic acid construct comprising the polynucleotide, preferably the nucleic acid construct is a vector.

The present invention also provides a host cell comprising said polynucleotide or said nucleic acid construct or vector, preferably said cell is a prokaryotic cell, eukaryotic cell, yeast cell, mammalian cell, E. coli cell or CHO cell, NS0 cell, Sp2 /0 cells or BHK cells.

The present invention also provides a pharmaceutical composition comprising the fusion protein and a pharmaceutically acceptable carrier.

The present invention also provides a method of treating a tumor or cancer comprising administering the fusion protein or pharmaceutical composition to a subject in need thereof.

The present invention also provides the use of said fusion protein, polynucleotide, nucleic acid construct or vector, or pharmaceutical composition in the manufacture of a medicament for the treatment or prevention of a tumor or cancer. Tumors or cancers include solid tumors or non-solid tumors.

The present invention also provides a diagnostic kit comprising the fusion protein.

The present invention also provides a method for producing a fusion protein, comprising culturing a host cell under conditions allowing expression of the nucleic acid construct, and recovering the fusion protein produced from the culture.

The high-body structural fusion protein provided in the present invention has the same expression and production advantages as IgG because the second binding domain is inserted in the hinge region, and can be smoothly applied to the existing IgG expression platform and purification platform, and this Significantly reduce development costs. Specifically, compared to the bispecific antibody constructed with the existing bispecific antibody platform, the bispecific antibody constructed with the high-body platform has a high expression level and there is no mismatch problem between the light and heavy chains, thereby significantly reducing antibody production costs. In addition, by inserting a second binding domain of a certain size in the two hinge regions, the binding activity of the Fab region of the fusion protein and stability of the protein are not affected, but also stability and a longer half-life are obtained. In particular, the binding activity of the second binding domain to the target binding site is significantly improved compared to the binding activity of the corresponding soluble native binding fragment monomer to the corresponding target, indicating that the receptor or ligand of the selected second binding domain is signal transduced. Form dimerization or multimerization structures in natural signaling pathways to activate or inhibit pathways. In addition, when the second binding domain is inserted into the hinge region, the spatial positional relationship in the hinge region promotes dimerization formation of the corresponding receptor or ligand or fragment thereof, thereby mimicking the native signaling pathway, resulting in more markedly improved binding An activity is generated such that the fusion protein has a greatly increased activating or inhibitory capacity relative to a soluble receptor or ligand or fragment thereof. In addition, effective synergistic effect can be achieved by flexibly selecting the target of receptor inhibition or receptor activation through rational combination of the two binding domains. The bispecific fusion protein provided in the present invention has a more significant disease therapeutic effect, particularly in effectively treating or controlling disease progression in various tumors and cancers, compared to the case of separately administering two target drugs or bispecific antibodies of the prior art. has

The fusion protein of the present invention has the same expression and production advantages as IgG, and does not affect the binding activity of the Fab region of the fusion protein, thereby further improving stability and obtaining a higher half-life. In addition, the binding activity of the second binding structural domain to the target binding site was significantly improved compared to the binding activity of the monomer to the target corresponding to the soluble native binding fragment.

1 is a schematic diagram of a high-body structural fusion protein.
2 shows inhibition curves of Hib-PDV and its control against mouse colon cancer model tumors.
3 shows inhibition curves of Hib-PDC and its control against mouse colon cancer tumors.
4 is a comparison chart of Hib-PLT and its control for promoting cell secretion of IFN-γ factor at a level of 10-100 nM.

Justice

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates. Those of ordinary skill in the art are skilled in the art in Dictionary of Cell and Molecular Biology, third edition, 1999, Academic Press; the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, second edition, 2002, CRC Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, revised edition, 2000 , Oxford University Press.

Amino acids relevant to the present invention may be represented by the commonly known three-letter symbols or single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Likewise, nucleotides may be represented by their commonly recognized one-letter codes.

In the present invention, the method for determining or numbering the complementarity determining region (CDR) of an antibody variable domain is IMGT, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda MD (1991)), Chothia, AbM, and contact methods.

For the purposes of the present invention, "homology", "identity" or "similarity" between two nucleic acid or amino acid sequences refers to the percentage of comparison between two sequences obtained after optimal alignment of identical nucleotide or amino acid residues.

The percentages are statistical only and the differences between the two sequences are randomly distributed and include the total length. Comparisons between two nucleic acid or amino acid sequences are generally performed by comparing these sequences after alignment in an optimal manner, and may be done through segments or "comparison windows". In addition to manual implementation, the local homology algorithm of Smith and Waterman (1981) was used or [Ad. App. Math. 2: 482], using the local homology algorithm of Neddleman and Wunsch (1970) or [J. MoI. Biol. 48: 443], using the similarity search method of Pearson and Lipman (1988) or [Proc. Natl. Acad. Sci. USA 85: 2444), and using computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or BLAST N or BLAST). Optimal alignment for sequence comparison can be performed through P comparison software).

As broadly defined, an immunoglobulin in the context of the present invention refers to an animal protein having antibody activity consisting of two identical light chains and two identical heavy chains. It is an important class of immune effector molecules, proteins produced by lymphocytes of the higher animal immune system, and can be transformed into antibodies by induction of antigens. Due to their different structures, they can be divided into five types: IgG, IgA, IgM, IgD and IgE. Preferably, the immunoglobulin according to the present invention is IgG.

As broadly defined, a receptor related to the present invention is a kind of specific protein that exists in a cell membrane or cell and is capable of activating a series of biochemical reactions in a cell by binding to a specific signaling molecule outside the cell, and the cell is activated under an external stimulus. to produce a corresponding effect. A biologically active substance that binds to a receptor is collectively called a ligand. Binding of receptors and ligands results in molecular conformational changes, which trigger cellular responses such as mediating intercellular signal transduction, intercellular adhesion, and endocytosis.

The bispecific fusion protein according to the present invention is based on the structure of an immunoglobulin having a second binding domain inserted in the hinge region. Thus, the bispecific fusion protein according to the present invention has two binding domains.

Example

The invention will be further illustrated by the following examples. It should be noted that the following examples are for further elaboration and explanation of the present invention and should not be construed as limiting the present invention.

Example 1. Construction of Proteins

In this example, the following protein molecules were prepared using conventional methods in the art, and transiently or stably expressed according to conventional methods in the art:

(1) three high body bispecific fusion proteins: Hib-PLT, Hib-PDC and Hib-PDV;

(2) Control protein 1-3: constructed according to the bifunctional molecular structure of WO2015/118175;

(3) Other control proteins: TGF-β-R-His, VEGF-R-His, CD80-His, PD-L1 control antibody.

Target combinations of fusion proteins Fusion protein code target of the first binding domain target of the second binding domain Inserting the second binding domain fragment peptide linker Hib-PLT PD-L1 TGF-β TGF-βRII extracellular domain fragment (GGGGS) ₃ Hib-PDC PD-1 CD28 CD80 (GGGGS) ₃ Hib-PDV PD-1 VEGF Combination of the second extracellular region of VEGFR1 and the third extracellular region of VEGFR2 (GGGGS) ₃ control protein 1 PD-L1 TGF-β TGF-βRII extracellular domain fragment (GGGGS) ₃ control protein 2 PD-1 CD28 CD80 (GGGGS) ₃ control protein 3 PD-1 VEGF Combination of the second extracellular region of VEGFR1 and the third extracellular region of VEGFR2 (GGGGS) ₃

The nucleic acid sequences and amino acid sequences of the heavy and light chains of the fusion protein Hib-PLT are as follows:

Nucleic acid sequence of the fusion protein Hib-PLT heavy chain (SEQ ID NO: 78):

ACCATCCCTCCACACGTGCAGAAGAGCGTGAACAATGACATGATCGTCACCGACAACAACGGCGCCGTCAAGTTCCCCCAGCTGTGCAAATTCTGCGACGTGAGGTTCTCCACGTGCGACAACCAGAAGAGCTGTATGAGCAACTGCAGCATCACATCCATCTGCGAAAAACCCCAGGAAGTGTGCGTCGCCGTCTGGCGGAAGAACGACGAGAACATCACACTGGAGACCGTGTGCCACGACCCCAAACTGCCCTACCACGACTTCATCCTGGAGGACGCCGCCAGCCCAAAGTGCATCATGAAAGAGAAGAAGAAGCCGGGCGAGACTTTCTTCATGTGCTCCTGCAGCTCCGACGAGTGCAACGATAATATCATCTTCAGCGAAGAATACAACACATCTAACCCAGAC

NOTE: The bold underlined sequence is the TGF-βRII nucleic acid sequence.

Amino acid sequence of the fusion protein Hib-PLT heavy chain (SEQ ID NO: 72):

QVQLQESGPG LVKPSETLSL TCTVSGFSLS RYSVHWIRQP PGKGLEWLGM IWGVGTTDYN 60

SALKSRLTIS KDTSKNQFSL KLSSVTAADT AVYYCARNWG TADYFDYWGQ GTTVTVSSAS 120

TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 180

YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKV EPKS CDK GGGGSGG GGSGGGGS TI 240

PPHVQKSVNN DMIVTDNNGA VKFPQLCKFC DVRFSTCDNQ KSCMSNCSIT SICEKPQEVC 300

VAVWRKNDEN ITLETVCHDP KLPYHDFILE DAASPKCIMK EKKKPGETFF MCSCSSDECN 360

DNIIFSEEYN TSNPD GGGGS GGGGSGGGGS THTCPPCPAP ELLGGP SVFL FPPKPKDTLM 420

ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD 480

WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 540

YPSIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL 600

HNHYTQKSLS LSPGK 615

NOTE: Bold and underlined fragments are hinge region sequences, peptide linkers are italics, and underlined and bolded sequences are TGF-βRII amino acid sequences.

Nucleic acid sequence of fusion protein Hib-PLT light chain (SEQ ID NO: 79):

GATATCGTGCTGACCCAGTCTCCAGCTTCCCTGGCCGTGTCCCCAGGACAGAGGGCCACCATCACATGTCGGGCTTCCAAGAGCGTGCACACAAGCGGCTACTCTTATATGCATTGGTACCAGCAGAAGCCCGGCCAGCCCCCTAAGCTGCTGATCTATCTGGCTTCCAACCTGGAGAGCGGAGTGCCAGCTAGGTTCTCTGGCTCCGGCAGCGGCACCGACTTTACCCTGACAATCAATCCTGTGGAGGCCAACGATACAGCTAATTACTATTGCCAGCACTCCGGAGAGCTGCCATACACCTTCGGCGGAGGCACAAAGGTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Amino acid sequence of fusion protein Hib-PLT light chain (SEQ ID NO: 73):

DIVLTQSPAS LAVSPGQRAT ITCRASKSVH TSGYSYMHWY QQKPGQPPKL LIYLASNLES 60

GVPARFSGSG SGTDFTLTIN PVEANDTANY YCQHSGELPY TFGGGTKVEI KRTVAAPSVF 120

IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS 180

STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC 218

The nucleic acid sequences and amino acid sequences of the heavy and light chains of the fusion protein Hib-PDC are as follows:

Nucleic acid sequence of fusion protein Hib-PDC heavy chain (SEQ ID NO: 74):

CAGGTGCAGCTCGTGGAGAGCGGGGGAGGCGTCGTCCAGCCTGGCCGTAGCCTGCGGCTGGACTGTAAGGCTAGCGGAATCACGTTCAGCAACAGCGGAATGCACTGGGTCAGACAGGCTCCTGGCAAAGGCCTCGAATGGGTCGCCGTGATCTGGTACGACGGGAGCAAGAGATACTACGCAGATTCAGTGAAGGGCCGTTTCACAATCAGCCGTGACAACTCGAAGAACACACTGTTCCTGCAGATGAACAGCCTGAGGGCAGAAGACACAGCAGTCTACTACTGCGCTACAAATGACGACTACTGGGGCCAGGGAACACTGGTCACCGTGAGCTCAGCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCGGTGGCGGCGGAAGCGGCGGCGGAGGCAGCGGCGGAGGCGGCAGC GTGATCCATGTCACGAAGGAGGTCAAAGAAGTGGCCACCCTCAGCTGCGGTCACAACGTCAGCGTGGAAGAGTTGGCCCAGACCAGAATCTACTGGCAGAAGGAGAAGAAGATGGTCTTGACCATGATGAGCGGCGACATGAACATCTGGCCAGAGTACAAGAATAGAACTATCTTCGACATCACCAATAACCTGAGCATCGTGATCCTCGCTTTGCGTCCGAGCGACGAAGGCACCTACGAGTGCGTAGTGCTAAAGTACGAGAAAGACGCCTTCAAGCGGGAGCACCTGGCAGAAGTAACCCTGAGCGTGAAGGCAGACTTCCCAACGCCTAGCATCTCCGACTTCGAGATCCCCACAAGCAACATCCGGCGGATCATCTGTAGCACCTCCGGGGGTTTCCCCGAGCCTCACCTGTCCTGGCTCGAGAACGGCGAGGAGCTGAACGCCATCAACACCACCGTGAGCCAGGACCCCGAGACAGAACTGTACGCCGTGAGCTCCAAGCTCGACTTCAACATGACCACAAATCACAGCTTCATGTGCCTCATCAAGTACGGACACCTGCGGGTGAATCAGACGTTCAACTGGAAC GGCGGGGGGGGTAGCGGCGGCGGGGGCTCAGGTGGGGGGGGCTCAAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATAG

NOTE: Underlined and bolded sequences are CD80 nucleic acid sequences.

Amino acid sequence of the fusion protein Hib-PDC heavy chain (SEQ ID NO: 68):

QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAV IWYDGSKRYY 60

ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT VSSASTKGPS 120

VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS 180

VVTVPSSSLG TKTYTCNVDH KPSNTKVDKR VES GGGGSGG GGSGGGGS VI HVTKEVKEVA 240

TLSCGHNVSV EELAQTRIYW QKEKKMVLTM MSGDMNIWPE YKNRTIFDIT NNLSIVILAL 300

RPSDEGTYEC VVLKYEKDAF KREHLAEVTL SVKADFPTPS ISDFEIPTSN IRRIICSTSG 360

GFPEPHLSWL ENGEELNAIN TTVSQDPETE LYAVSSKLDF NMTTNHSFMC LIKYGHLRVN 420

QTFNWN GGGG SGGGGSGGGG S KYGPPCPPC PAPEFLGGPS VFLFPPKPKD TLMISRTPEV 480

TCVVVDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST YRVVSVLTVL HQDWLNGKEY 540

KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV 600

EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK 660

SLSLSLGK 668

NOTE: Peptide linkers are italicized and underlined and bold are CD80 amino acid sequences.

Nucleic acid sequence of fusion protein Hib-PDC light chain (SEQ ID NO: 75):

GAGATCGTGCTGACACAGAGCCCAGCCACTCTGTCACTGTCCCCAGGAGAAAGGGCTACTCTGTCTTGCCGGGCAAGCCAGTCTGTCTCCAGCTACCTGGCCTGGTATCAGCAGAAGCCCGGACAGGCTCCTAGACTGCTGATCTACGACGCAAGTAACAGAGCCACCGGCATCCCCGCACGCTTCAGTGGCTCAGGCTCCGGAACAGACTTTACTCTGACCATCTCTAGTCTGGAGCCTGAAGATTTCGCCGTGTACTATTGTCAGCAGAGCTCTAATTGGCCTAGAACCTTCGGCCAGGGCACCAAAGTCGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Amino acid sequence of fusion protein Hib-PDC light chain (SEQ ID NO: 69):

EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60

RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIKRTV AAPSVFIFPP 120

SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180

LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214

The nucleic acid sequences and amino acid sequences of the heavy and light chains of the fusion protein Hib-PDV are as follows:

Nucleic acid sequence of fusion protein Hib-PDV heavy chain (SEQ ID NO: 76):

CAGGTGCAGCTCGTGGAGAGCGGGGGAGGCGTCGTCCAGCCTGGCCGTAGCCTGCGGCTGGACTGTAAGGCTAGCGGAATCACGTTCAGCAACAGCGGAATGCACTGGGTCAGACAGGCTCCTGGCAAAGGCCTCGAATGGGTCGCCGTGATCTGGTACGACGGGAGCAAGAGATACTACGCAGATTCAGTGAAGGGCCGTTTCACAATCAGCCGTGACAACTCGAAGAACACACTGTTCCTGCAGATGAACAGCCTGAGGGCAGAAGACACAGCAGTCTACTACTGCGCTACAAATGACGACTACTGGGGCCAGGGAACACTGGTCACCGTGAGCTCAGCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCGGTGGCGGCGGAAGCGGCGGCGGAGGCAGCGGCGGAGGCGGCAGC GGTAGACCATTCGTAGAGATGTACAGCGAAATCCCCGAAATCATCCACATGACTGAAGGAAGGGAGCTCGTCATCCCCTGCCGGGTTACGTCACCTAACATCACTGTTACTCTGAAGAAGTTCCCACTCGACACTTTGATCCCTGATGGAAAACGCATCATCTGGGACAGCAGAAAGGGCTTCATCATCTCAAATGCAACGTACAAAGAAATCGGACTCCTGACCTGTGAAGCAACAGTCAATGGACATTTGTATAAGACAAACTATCTCACACATCGACAGACCAATACAATCATCGATGTGGTTCTGAGCCCGTCTCATGGAATCGAACTATCTGTTGGAGAAAAGCTCGTCCTGAATTGTACAGCAAGAACTGAACTGAATGTGGGAATCGACTTCAACTGGGAATACCCTTCTTCGAAGCATCAGCATAAGAAACTCGTAAACCGAGACCTAAAGACCCAGTCTGGGAGCGAGATGAAGAAATTCTTGAGCACCCTGACTATCGATGGTGTAACCCGGAGCGACCAGGGATTGTACACCTGTGCAGCATCCAGCGGGCTGATGACCAAGAAGAACAGCACATTCGTCAGGGTCCATGAACCC GGCGGGGGGGGTAGCGGCGGCGGGGGCTCAGGTGGGGGGGGCTCAAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATAG

NOTE: Underlined and bold font are VEGFR nucleic acid sequences.

Amino acid sequence of fusion protein Hib-PDV heavy chain (SEQ ID NO: 70):

QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAV IWYDGSKRYY 60

ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT VSSASTKGPS 120

VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS 180

VVTVPSSSLG TKTYTCNVDH KPSNTKVDKR VES GGGGSGG GGSGGGGS GR PFVEMYSEIP 240

EIIHMTEGRE LVIPCRVTSP NITVTLKKFP LDTLIPDGKR IIWDSRKGFI ISNATYKEIG 300

LLTCEATVNG HLYKTNYLTH RQTNTIIDVV LSPSHGIELS VGEKLVLNCT ARTELNVGID 360

FNWEYPSSKH QHKKLVNRDL KTQSGSEMKK FLSTLTIDGV TRSDQGLYTC AASSGLMTKK 420

NSTFVRVHEP GGGGSGGGGS GGGGS KYGPP CPPCPAPEFL GGPSVFLFPP KPKDTLMISR 480

TPEVTCVVVD VSQEDPEVQF NWYVDGVEVH NAKTKPREEQ FNSTYRVVSV LTVLHQDWLN 540

GKEYKCKVSN KGLPSSIEKT ISKAKGQPRE PQVYTLPPSQ EEMTKNQVSL TCLVKGFYPS 600

DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSRLTVDKS RWQEGNVFSC SVMHEALHNH 660

YTQKSLSLSL GK 672

NOTE: Peptide linkers are italicized and underlined and bold are VEGF amino acid sequences.

Nucleic acid sequence of fusion protein Hib-PDV light chain (SEQ ID NO: 77):

GAGATCGTGCTGACACAGAGCCCAGCCACTCTGTCACTGTCCCCAGGAGAAAGGGCTACTCTGTCTTGCCGGGCAAGCCAGTCTGTCTCCAGCTACCTGGCCTGGTATCAGCAGAAGCCCGGACAGGCTCCTAGACTGCTGATCTACGACGCAAGTAACAGAGCCACCGGCATCCCCGCACGCTTCAGTGGCTCAGGCTCCGGAACAGACTTTACTCTGACCATCTCTAGTCTGGAGCCTGAAGATTTCGCCGTGTACTATTGTCAGCAGAGCTCTAATTGGCCTAGAACCTTCGGCCAGGGCACCAAAGTCGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Amino acid sequence of fusion protein Hib-PDV light chain (SEQ ID NO: 71):

EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60

RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIKRTV AAPSVFIFPP 120

SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180

LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214

Amino acid sequences of heavy and light chains of control protein 1

Amino acid sequence of control protein 1 heavy chain (SEQ ID NO: 124):

QVQLQESGPG LVKPSETLSL TCTVSGFSLS RYSVHWIRQP PGKGLEWLGM IWGVGTTDYN 60

SALKSRLTIS KDTSKNQFSL KLSSVTAADT AVYYCARNWG TADYFDYWGQ GTTVTVSSAS 120

TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 180

YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS 240

VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 300

YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT 360

KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 420

GNVFSCSVMH EALHNHYTQK SLSLSPGK GG GGSGGGGSGG GGS TIPPHVQ KSVNNDMIVT 480

DNNGAVKFPQ LCKFCDVRFS TCDNQKSCMS NCSITSICEK PQEVCVAVWR KNDENITLET 540

VCHDPKLPYH DFILEDAASP KCIMKEKKKP GETFFMCSCS SDECNDNIIF SEEYNTSNPD 600

NOTE: Peptide linkers are italicized and underlined and bold are TGF-βRII amino acid sequences.

Amino acid sequence of control protein 1 light chain (SEQ ID NO: 125):

DIVLTQSPAS LAVSPGQRAT ITCRASKSVH TSGYSYMHWY QQKPGQPPKL LIYLASNLES 60

GVPARFSGSG SGTDFTLTIN PVEANDTANY YCQHSGELPY TFGGGTKVEI KRTVAAPSVF 120

IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS 180

STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC 218

Amino acid sequences of heavy and light chains of control protein 2

Amino acid sequence of control protein 2 heavy chain (SEQ ID NO: 126):

QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAV IWYDGSKRYY 60

ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT VSSASTKGPS 120

VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS 180

VVTVPSSSLG TKTYTCNVDH KPSNTKVDKR VESKYGPPCP PCPAPEFLGG PSVFLFPPKP 240

KDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT 300

VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC 360

LVKGFYPSD AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV 420

MHEALHNHYT QKSLSLSLGK GGGGSGGGGS GGGGS VIHVT KEVKEVATLS CGHNVSVEEL 480

AQTRIYWQKE KKMVLTMMSG DMNIWPEYKN RTIFDITNNL SIVILALRPS DEGTYECVVL 540

KYEKDAFKRE HLAEVTLSVK ADFPTPSISD FEIPTSNIRR IICSTSGGFP EPHLSWLENG 600

EELNAINTTV SQDPETELYA VSSKLDFNMT TNHSFMCLIK YGHLRVNQTF NWN 653

NOTE: Peptide linkers are italicized and underlined and bold are CD80 amino acid sequences.

Amino acid sequence of control protein 2 light chain (SEQ ID NO: 127):

EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60

RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIKRTV AAPSVFIFPP 120

SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180

LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214

Amino acid sequences of heavy and light chains of control protein 3

Amino acid sequence of control protein 3 heavy chain (SEQ ID NO: 128):

QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAV IWYDGSKRYY 60

ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT VSSASTKGPS 120

VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS 180

VVTVPSSSLG TKTYTCNVDH KPSNTKVDKR VESKYGPPCP PCPAPEFLGG PSVFLFPPKP 240

KDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT 300

VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC 360

LVKGFYPSD AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV 420

MHEALHNHYT QKSLSLSLGK GGGGSGGGGS GGGGS GRPFV EMYSEIPEII HMTEGRELVI 480

PCRVTSPNIT VTLKKFPLDT LIPDGKRIIW DSRKGFIISN ATYKEIGLLT CEATVNGHLY 540

KTNYLTHRQT NTIIDVVLSP SHGIELSVGE KLVLNCTART ELNVGIDFNW EYPSSKHQHK 600

KLVNRDLKTQ SGSEMKKFLS TLTIDGVTRS DQGLYTCAAS SGLMTKKNST FVRVHEP 657

NOTE: Peptide linkers are italicized and underlined and bold are VEGFR amino acid sequences.

Amino acid sequence of control protein 3 light chain (SEQ ID NO: 129):

EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60

RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIKRTV AAPSVFIFPP 120

SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180

LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214

TGF-β-R-His

TGF-β-R-His is a His-tagged TGF-βRII extracellular region fragment, wherein the amino acid sequence of the TGF-βRII extracellular region fragment (SEQ ID NO: 137) is as follows:

TIPPHVQKSV NNDMIVTDNN GAVKFPQLCK FCDVRFSTCD NQKSCMSNCS ITSICEKPQE 60

VCVAVWRKND ENITLETVCH DPKLPYHDFI LEDAASPKCI MKEKKKPGET FFMCSCSSDE 120

CNDNIIFSEE YNTSNPD 137

VEGF-R-His amino acid sequence

VEGF-R-His is a His-tagged combination fragment of the second extracellular region of VEGFR1 and the third extracellular region of VEGFR2, wherein the combination fragment of the second extracellular region of VEGFR1 and the third extracellular region of VEGFR2 The amino acid sequence (SEQ ID NO: 130) is as follows:

GRPFVEMYSE IPEIIHMTEG RELVIPCRVT SPNITVTLKK FPLDTLIPDG KRIIWDSRKG 60

FIISNATYKE IGLLTCEATV NGHLYKTNYL THRQTNTIID VVLSPSHGIE LSVGEKLVLN 120

CTARTELNVG IDFNWEYPSS KHQHKKLVNR DLKTQSGSEM KKFLSTLTID GVTRSDQGLY 180

TCAASSGLMT KKNSTFVRVH EP 202

CD80-His amino acid sequence

CD80-His is a His-tagged CD80 fragment, wherein the amino acid sequence of the CD80 fragment (SEQ ID NO: 32) is as follows:

VIHVTKEVKE VATLSCGHNV SVEELAQTRI YWQKEKKMVL TMMSGDMNIW PEYKNRTIFD 60

ITNNLSIVIL ALRPSDEGTY ECVVLKYEKD AFKREHLAEV TLSVKADFPT PSISDFEIPT 120

SNIRRIICST SGGFPEPHLS WLENGEELNA INTTVSQDPE TELYAVSSKL DFNMTTNHSF 180

MCLIKYGHLR VNQTFNWN 198

Example 2. CHO expression

A. Cell thawing and expansion:

CHO host cells were thawed in a culture volume of 10 mL and culture conditions of 37.0° C., 200 rpm, 8% CO ₂ and 80% humidity. After thawing, the cell density was 2.82 x 10 ⁵ cells/mL and the viability was 95.41%.

After 72 hours of cell expansion and culture, the density of viable cells was 2.22 x 10 ⁶ cells/mL, and the viability was 98.32%.

For cell re-injection operations, cells were re-injected at 5 x 10 ⁵ cells/mL. After continuous culture for 48 hours, the density of viable cells was 2Х10 ⁶ cells/mL and the viability was 98.68%.

Cells were seeded at 5 x 10 ⁵ cells/well in a 6-well plate at a culture volume of 2 mL/well, placed in a carbon dioxide incubator (culture conditions: 37° C., 8% CO ₂ ), for engraftment and transfection on the next day. kept overnight.

B. Transfection

As the transfection reagent, Lipofectamine® LTX reagent (Invitrogen, catalog number 15338-100) containing LTX and Plus reagent was used.

Transfection procedure: medium change in 6-well plate and transfection complex preparation: LTX: 9 μl/well + plasmid: 2.5 μg/well + plus: 2.5 μl/well. Transfection complexes were added at 250 μl/well in 6 well plates. The medium in each well of the 6-well plate was replaced 5 hours after cell transfection. 48 hours after transfection, the supernatant was taken and the antibody content of the supernatant was detected by a double antigen ELISA method.

C. Batch and Fed-Batch Cultures

Forty-eight hours after transfection, cells in 6-well plates were digested, collected in cell culture tubes, and adapted to grow in suspension at a cell density of 3×10 ⁵ cells/mL. When the cell viability recovered to about 90%, the Hib-PLT expressing cell pool and the control protein 1 expressing cell pool were cultured in batch culture, and Hib-PDC, control protein 2, Hib-PDV and control protein 3 were fed-batch cultured.

Batch culture: Cells were cultured in TPP cell culture tubes at a cell density of 3 x 10 ⁵ cells/mL, a volume of 30 mL, and culture conditions at 37.0 °C, 200 rpm, 8% CO ₂ and 80% humidity, and cell viability and metabolism was monitored daily. The culture was terminated when the viability reached <60%, and the cell supernatant was collected after completion of the culture to detect the concentration.

Fed-batch culture: Cells are cultured in TPP cell culture tubes at a cell density of 3 x 10 ⁵ cells/mL, a volume of 30 mL, and culture conditions at 37.0 °C, 200 rpm, 8% CO ₂ and 80% humidity. cultured. Cell-enriched medium was provided according to a specific fed-batch scheme (see Table 5) while Glc was regulated to 2-6 g/L. The culture was terminated until the viability became <60%, and the cell supernatant was collected after the completion of the culture to detect the concentration.

Fed-Batch Plan 0 days 3Х10 ⁵ /ml, culture volume 30 ml 4、6、8、10、12 days 900 μL CellBoost7A、90 μL CellBoost7B 16 days VCD and viability detection, sample stockpiling and end of experiment

Table 6 shows the expression level results of the hibody bispecific fusion proteins Hib-PLT, Hib-PDC and Hib-PDV and their corresponding control proteins. These results show that the relative value of the increase in Hib-PLT expression level was increased by 52.85% compared to the corresponding control protein; The relative value of the increase in Hib-PDC expression level was increased by 33.43% compared to the corresponding control protein; The relative value of the increase in Hib-PDV expression level is increased by 25.6% compared to the corresponding control protein, indicating that the expression level of the hi-body bispecific fusion protein provided in the present invention is significantly superior to the expression level of the corresponding control protein, It can be seen that the bispecific antibody provided in the present invention has an unexpected technical effect.

Expression levels of bispecific antibodies category Culture method expression level increase relative value Hib-PLT batch culture 188 mg/L 52.85% control protein 1 batch culture 123 mg/L Hib-PDC fed-batch culture 349.59 mg/L 33.43% control protein 2 fed-batch culture 262 mg/L Hib-PDV fed-batch culture 243.95 mg/L 25.6% control protein 3 fed-batch culture 194.22 mg/L

NOTE: Relative value of increase = (expression level of the bispecific fusion protein provided in the present invention - expression level of the corresponding control protein) / the expression level of the corresponding control protein.

Example 3. Measurement of binding activity

1. Measurement of Hib-PLT binding activity:

(1) Coating antigen: TGF-β1 was diluted to 2 μg/ml with coating buffer, added to 96-well plate at 100 μl/well, and left at 2-8° C. overnight;

(2) Plate washing: The plate was automatically washed 3 times at 350 μl/well with PBST used as an eluent, washed and dried by tapping lightly.

(3) Block plate: Block the plate at 300 μl/well with 3% BSA-PBST using as blocking solution and incubate at 37 °C for 2 h.

(4) Washing the plate: The plate was automatically washed 3 times at 350 μl/well with PBST used as the eluent, and dried by tapping lightly after washing.

(5) Addition of sample: Each concentration point of the sample is as follows. Samples from each concentration point were sequentially added to a 96-well plate at 100 μl/well, each concentration point paralleled to two replicate wells and the plate was incubated at 37 °C for 2 h.

experimental group S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11 Hib-PLT 555.556 55.556 5.556 1.852 0.617 0.206 0.069 0.023 0.008 0.001 0.000 TGF-β-R-His 64516.000 6451.600 645.160 215.053 71.684 23.895 7.965 2.655 0.885 0.088 0.009 (Unit: nM)

(6) Plate washing: The plate was automatically washed 3 times at 350 μl/well with PBST used as the eluent, washed and dried by tapping lightly.

(7) Addition of secondary antibody:

Hib-PLT sample wells: goat-anti-human-Fc-HRP was diluted 1:5000 and added at 100 μl/well. The plates were then incubated at 37 °C for 2 h.

TGF-β-R-His wells: Anti-His-HRP was diluted 1:5000 and added at 100 μl/well. The plates were then incubated at 37 °C for 2 h.

(8) Plate washing: The plate was automatically washed 5 times at 350 μl/well with PBST used as the eluent, washed and dried by tapping lightly.

(9) Color development: The plate was developed for 2 minutes at 100 μl/well using TMB used as a color development solution.

(10) End of color development: Plates were terminated for color development at 50 μl/well with 2M H ₂ SO ₄ used as stop solution.

(11) Reading: Absorbance values were read at 450/655 nm respectively using an MD microplate reader. A plot was created to draw a 4-PL curve with the concentration of the test sample as the horizontal axis and the absorbance value as the vertical axis, and the software provided the EC50 values automatically.

2. Measurement of Hib-PDV Binding Activity:

(1) Coating antigen: VEGF was diluted to 1 μg/ml with coating buffer, added to 96-well plate at 100 μl/well, and left overnight at 2-8° C.;

(2) Plate washing: The plate was automatically washed 3 times at 350 μl/well with PBST used as the eluent, washed and dried by tapping lightly.

(3) Block plate: Block the plate at 300 μl/well with 3% BSA-PBST using as blocking solution and incubate at 37 °C for 2 h.

(4) Washing the plate: The plate was automatically washed 3 times at 350 μl/well with PBST used as the eluent, washed and dried by tapping lightly.

(5) Addition of sample: Each concentration point of the sample is as follows. Samples from each concentration point were sequentially added to a 96-well plate at 100 μl/well, each concentration point paralleled to two replicate wells and the plate was incubated at 37 °C for 2 h.

experimental group S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11 Hib-PDV 555.556 55.556 5.556 1.852 0.617 0.206 0.069 0.023 0.005 0.001 0.000 VEGFR-His 43478.300 4347.830 434.783 144.928 48.309 16.103 5.368 1.789 0.358 0.036 0.004 (Unit: nM)

(6) Plate washing: Plates were automatically washed 3 times at 350 μl/well with PBST used as eluent, washed and dried by tapping lightly.

(7) Addition of secondary antibody:

Hib-PDV sample wells: Goat-anti-human-Fc-HRP was diluted 1:5000 and added at 100 μl/well. The plates were then incubated at 37 °C for 2 h.

VEGFR-His wells: Anti-His-HRP was diluted 1:5000 and added at 100 μl/well. The plates were then incubated at 37 °C for 2 h.

(8) Plate washing: The plate was automatically washed 5 times at 350 μl/well with PBST used as the eluent, washed and dried by tapping lightly.

(9) Color development: Plates were developed for 2 minutes at 100 μl/well using TMB used as a chromogenic solution.

(10) End of color development: Plates were terminated for color development at 50 μl/well with 2M H ₂ SO ⁴ used as stop solution.

(11) Reading: Absorbance values were read at 450/655 nm respectively using an MD microplate reader. A plot was created to draw a 4-PL curve with the concentration of the test sample as the horizontal axis and the absorbance value as the vertical axis, and the software provided the EC50 values automatically.

3. Measurement of Hib-PDC Binding Activity:

(1) Coating antigen: CTLA-4 protein was re-dissolved in 1 mL of sterile water at a concentration of 200 μg/mL, diluted to 20 μg/ml with coating buffer, added to a 96-well plate at 100 μl/well, and then at 2-8°C. left overnight;

(2) Plate washing: The plate was automatically washed 3 times at 350 μl/well with PBST used as the eluent, washed and dried by tapping lightly.

(3) Plate blocking: Plates were blocked at 300 μl/well with 3% BSA-PBST using as blocking solution and incubated at 37 °C for 2 h ± 20 min.

(4) Washing the plate: The plate was automatically washed 3 times at 350 μl/well with PBST used as the eluent, washed and dried by tapping lightly.

(5) Sample addition: Biotinylated samples were diluted with PBST. Each concentration point of the sample is as follows. Samples from each concentration point were sequentially added to a 96-well plate at 100 μl/well, each concentration point parallel to two replicate wells, blank wells were PBST, and plates were incubated at 37 °C for 2 h ± 20 min. incubated.

experimental group S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 Hib-PDC 1000000 100000 10000 3333.333 1111.111 370.370 123.457 41.152 13.717 1.372 CD80-His 1000000 100000 10000 3333.333 1111.111 370.370 123.457 41.152 13.717 1.372 (Unit: nM)

(6) Plate washing: Plates were automatically washed 3 times at 350 μl/well with PBST used as eluent, washed and dried by tapping lightly.

(7) Addition of secondary antibody:

Hib-PDC sample wells and CD80-His wells: Streptavidin-HRP was diluted 1:2000 and added at 100 μl/well. The plates were then incubated at 37 °C for 1 h ± 10 min.

(8) Plate washing: The plate was automatically washed 5 times at 350 μl/well with PBST used as the eluent, washed and dried by tapping lightly.

(9) Color development: 100 μl/well of TMB Double Slow substrate was added and the plate was allowed to develop color at room temperature shaded from light for 6 minutes.

(10) End of color development: Plates were terminated for color development at 50 μl/well with 2M H ₂ SO ₄ used as stop solution.

(11) Reading: Absorbance values were read at 450/655 nm respectively using an MD microplate reader. A plot was created to draw a 4-PL curve with the concentration of the test sample as the horizontal axis and the absorbance value as the vertical axis, and the software provided the EC50 values automatically. (See Table 10).

EC50 values for each test group experimental group EC50 value Hib-PLT 0.164 nM TGF-β-R-His 11.60 nM Hib-PDV 0.061 nM VEGFR-His 2154 nM Hib-PDC 24.36 nM CD80-His 50.69 nM

As a result of measurement of binding activity, as a result of comparing Hib-PLT and TGF-β receptor monomer, the EC50 value of Hib-PLT was 0.164 nM, the EC50 value of the TGF-β receptor monomer was 11.60 nM, and the binding activity of Hib-PLT was was about 70 times better than the binding activity of the TGF-β receptor monomer. As a result of comparison between Hib-PDV and VEGF receptor monomer, Hib-PDV has an EC50 value of 0.061 nM, VEGF receptor monomer has an EC50 value of 2154 nM, and Hib-PDV binding activity is about 35,311 times greater than that of the VEGF receptor monomer. was better. As a result of comparison between Hib-PDC and CD80 monomer, the EC50 value of Hib-PDC was 24.36 nM, the EC50 value of CD80 monomer was 50.69 nM, and the binding activity of Hib-PDC was about 2 times superior to that of CD80 monomer. That is, the binding activity of Hib-PLT, Hib-PDV, and Hib-PDC constructed with the diabody model provided in the present invention was much superior to that of the corresponding monomers.

Example 4. Efficacy of Hib-PDV on Subcutaneously Transplanted Tumors of Colon Cancer MC38 Cells in C57BL/6J-hPD-1 Mice

Thirty-two 7-week-old, C57BL/6J humanized PD-1 transgenic mice (Shanghai Model Organisms Center, Inc.) were collected. 0.1 mL of colon cancer MC38 cell suspension (1 x 10 ⁶ cells/mouse) was inoculated subcutaneously into the right flank of mice. When the tumors grew to about 100 mm ³ , mice were randomly assigned to vehicle (PBS) group, Hib-PDV (1.25 mg/kg) group, Hib-PDV (2.5 mg/kg) group and Hib-PDV group according to tumor size. (5 mg/kg) groups were divided into 4 groups each, and each group had 8 tumor-bearing mice. It was administered intraperitoneally once a week and administered a total of 3 times, and the major and minor axis and body weight of the tumor were measured twice a week during the administration period. On day 21 after administration, the tumor growth inhibition rate (TGI _RTV ) was calculated as shown in Table 11 for drug efficacy evaluation.

Hib-PDV administration regimen for tumor suppressive activity in mouse colon cancer group administration group Volume
(mg/kg) route of administration dosing cycle Dosage One vehicle -- i.p. QWХ3 10 mL/kg 2 Hib-PDV 5 i.p. QWХ3 10 mL/kg 3 Hib-PDV 2.5 i.p. QWХ3 10 mL/kg 4 Hib-PDV 1.25 i.p. QWХ3 10 mL/kg

Calculation formula:

Tumor volume: TV = D ₁ ХD ₂ ² /2, where D1 and D2 represent the major and minor diameters of the tumor, respectively;

Relative tumor volume: RTV = T _VT /T _V1 , where T _V1 is the pre-dose tumor volume and T _VT is the tumor volume at each measurement;

Relative tumor inhibition rate: TGI _RTV (%) = (1-T _RTV /C _RTV ) x 100%, where T _RTV represents the RTV of the administration group or the positive control group, and C _RTV represents the RTV of the negative control group.

Results and Conclusions: The tumor suppressive effect of Hib-PDV on the mouse colon cancer model is shown in Table 12 and FIG. 2, and Table 12 shows the tumor volume and tumor growth inhibition rate of Hib-PDV on the mouse colon cancer model, and FIG. Graph of tumor growth after administration. Experimental results show that Hib-PDV has a significant inhibitory effect on mouse colon cancer model tumors.

Effect of Hib-PDV on Tumor Volume and Tumor Suppression in a Mouse Colon Cancer Model (Mean+SEM) administration group Tumor volume (TV, mm) ³ ) TGIRTV (%) D0 D21 vehicle 86+10 1299+174 -- Hib-PDV (5 mg/kg) 86+10 675+218 46 Hib-PDV (2.5 mg/kg) 86+8 755+305 47 Hib-PDV (1.25 mg/kg) 85+8 1088+259 14

Example 5. Efficacy of Hib-PDC on subcutaneously implanted tumors of transgenic hPD-L1 MC38 cells in C57BL/6J-hPD-1 mice

Thirty-two 7-9 week old humanized C57BL/6J humanized PD-1 transgenic female mice (Shanghai Model Organisms Center, Inc.) were collected. 0.1 mL of transgenic hPD-L1 MC38 cell suspension (1 x 10 ⁶ cells/mouse) was implanted subcutaneously into the back of the right forelimb of mice. When the tumors grew to about 100 mm ³ , mice were randomly assigned to vehicle (PBS) group, Hib-PDC (1.25 mg/kg) group, Hib-PDC (2.5 mg/kg) group and Hib-PDC group according to tumor size. (5 mg/kg) groups were divided into 4 groups each, and each group had 8 tumor-bearing mice. It was administered intraperitoneally once a week and administered a total of 3 times, and the major and minor axis and body weight of the tumor were measured twice a week during the administration period. On day 21 after administration, the tumor growth inhibition rate (TGI _RTV ) was calculated as shown in Table 13 for drug efficacy evaluation.

Dosing regimen of Hib-PDC for tumor suppressive activity of mouse colon cancer group administration group Dosage (mg/kg) route of administration Dosage dosing cycle One Vehicle (PBS) -- i.p. 10 ml/kg QWХ3 2 Hib-PDC 1.25 i.p. 10 ml/kg QWХ3 3 Hib-PDC 2.5 i.p. 10 ml/kg QWХ3 4 Hib-PDC 5 i.p. 10 ml/kg QWХ3

Calculation formula:

Tumor volume: TV = D ₁ ХD ₂ ² /2, where D1 and D2 represent the major and minor diameters of the tumor, respectively;

Relative tumor volume: RTV = T _VT /T _V1 , where T _V1 is the pre-dose tumor volume and T _VT is the tumor volume at each measurement;

Relative tumor inhibition rate: TGI _RTV (%) = (1-T _RTV /C _RTV ) x 100%, where T _RTV represents the RTV of the administration group or the positive control group, and C _RTV represents the RTV of the negative control group.

Results and Conclusions: The tumor suppressive effect of Hib-PDC on the mouse colon cancer model is shown in Table 14 and FIG. 3, and Table 14 shows the tumor volume and the tumor growth inhibition rate of Hib-PDC on the mouse colon cancer model, and FIG. Graph of tumor growth after administration. Experimental results show that Hib-PDC has a significant inhibitory effect on subcutaneously transplanted colorectal cancer tumors in mice.

Effect of Hib-PDC on Tumor Volume and Tumor Suppression in a Mouse Colon Cancer Model (Mean+SEM) army Volume
(mg/kg) Tumor volume (TV, mm) ³ ) TGIRTV (%) D0 D21 PBS -- 101±10 2319±517 -- Hib-PDC 1.25 101±8 1577±211 31 Hib-PDC 2.5 101±9 813±74 63 Hib-PDC 5 101±9 541±179 77

Example 6. Effect of Hib-PLT on Cytokine Secretion in Mixed Response of Allogeneic Lymphocytes

To evaluate the role of Hib-PLT in activating CD4 ⁺ T cells, an ELISA method was used to detect the concentration of the cytokine IFN-γ in the cell supernatant. Mononuclear lymphocytes from donor 1 were induced into mature dendritic cells with an in vitro DC cell rapid maturation in vitro kit (cat: CT-004, StemEry), and cells were collected after 48 hours and diluted to 2×10 ⁵ cells/mL. Donor 2 CD4 ⁺ T cells were enriched with magnetic beads (EasySep® Human CD4 ⁺ T Cell Enrichment Kit, StemCell, cat: 19052) and diluted to 1 x 10 ⁶ cells/mL. Mature dendritic cells and CD4 ⁺ T cells were mixed at a volume ratio of 1:1, and the mixed cell suspension was added to a 96-well plate at a volume of 100 μL/well. Hib-PLT, PD-L1 control antibody + TGF-β Trap (concomitant drug), PD-L1 control antibody, TGF-β Trap and human IgG1 (Biolegend) were mixed in a 96-well plate containing 0.01-100 nM of mixed cell suspension. was added to a final concentration of Plates were then placed in a 37 °C, 5% CO ₂ incubator for 120 h, centrifuged at 2000 rpm for 5 min, and 150-200 µL of cell supernatant was collected. IFN-γ factor was detected in the mixed reaction supernatants of allogeneic lymphocytes using an ELISA kit (Human IL-2 Quantikine ELISA Kit, R&D, cat: S2050; Human IFN-gamma Quantikine ELISA Kit, R&D, cat: SIF50C). One-way ANOVA was used to analyze the differences between groups.

Results and Conclusions: As shown in FIG. 4 , the level of Hib-PLT at a concentration of 10-100 nM that promotes the cellular secretion of IFN-γ factor was more significant than the group administered with the PD-L1 control antibody and TGF-β trap. very high (p<0.05). These results show that at a concentration level of 10-100 nM, Hib-PLT is superior to the combination drug group in promoting the activation of CD4 ⁺ T cells in the mixed lymphocyte response.

The invention has been illustrated by way of various specific examples. However, those of ordinary skill in the art may not limit the present invention to each specific embodiment, and those skilled in the art may make various changes or modifications within the scope of the present invention, Each of the technical features mentioned herein may be combined with each other without departing from the spirit and scope of the present invention. Such changes and modifications are within the scope of the present invention.

<110> RemeGen Co., Ltd. <120> BISPECIFIC FUSION PROTEIN AND APPLICATION THEREOF <130> PI22009 <150> CN 202010199036.2 <151> 2020-03-20 <160> 137 <170> KoPatentIn 3.0 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR1 <400> 1 Gly Phe Ser Leu Ser Arg Tyr 1 5 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR1 <400> 2 Gly Phe Ser Leu Ser Arg Tyr Ser Val His 1 5 10 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR1 <400> 3 Arg Tyr Ser Val His 1 5 <210> 4 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR1 <400> 4 Ser Arg Tyr Ser Val His 1 5 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR1 <400> 5 Gly Phe Ser Leu Ser Arg Tyr Ser 1 5 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR2 <400> 6 Trp Gly Val Gly Thr 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR2 <400> 7 Met Ile Trp Gly Val Gly Thr Thr Asp 1 5 <210> 8 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR2 <400> 8 Met Ile Trp Gly Val Gly Thr Thr Asp Tyr Asn Ser Ala Leu Lys Ser 1 5 10 15 <210> 9 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR2 <400> 9 Trp Leu Gly Met Ile Trp Gly Val Gly Thr Thr Asp 1 5 10 <210> 10 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR2 <400> 10 Ile Trp Gly Val Gly Thr Thr 1 5 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR3 <400> 11 Asn Trp Gly Thr Ala Asp Tyr Phe Asp Tyr 1 5 10 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR3 <400> 12 Asn Trp Gly Thr Ala Asp Tyr Phe Asp Tyr 1 5 10 <210> 13 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR3 <400> 13 Asn Trp Gly Thr Ala Asp Tyr Phe Asp Tyr 1 5 10 <210> 14 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR3 <400> 14 Ala Arg Asn Trp Gly Thr Ala Asp Tyr Phe Asp 1 5 10 <210> 15 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR3 <400> 15 Ala Arg Asn Trp Gly Thr Ala Asp Tyr Phe Asp Tyr 1 5 10 <210> 16 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR1 <400> 16 Arg Ala Ser Lys Ser Val His Thr Ser Gly Tyr Ser Tyr Met His 1 5 10 15 <210> 17 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR1 <400> 17 Arg Ala Ser Lys Ser Val His Thr Ser Gly Tyr Ser Tyr Met His 1 5 10 15 <210> 18 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR1 <400> 18 Arg Ala Ser Lys Ser Val His Thr Ser Gly Tyr Ser Tyr Met His 1 5 10 15 <210> 19 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR1 <400> 19 His Thr Ser Gly Tyr Ser Tyr Met His Trp Tyr 1 5 10 <210> 20 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR1 <400> 20 Lys Ser Val His Thr Ser Gly Tyr Ser Tyr 1 5 10 <210> 21 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR2 <400> 21 Leu Ala Ser Asn Leu Glu Ser 1 5 <210> 22 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR2 <400> 22 Leu Ala Ser Asn Leu Glu Ser 1 5 <210> 23 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR2 <400> 23 Leu Ala Ser Asn Leu Glu Ser 1 5 <210> 24 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR2 <400> 24 Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu 1 5 10 <210> 25 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR2 <400> 25 Leu Ala Ser One <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR3 <400> 26 Gln His Ser Gly Glu Leu Pro Tyr Thr 1 5 <210> 27 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR3 <400> 27 Gln His Ser Gly Glu Leu Pro Tyr Thr 1 5 <210> 28 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR3 <400> 28 Gln His Ser Gly Glu Leu Pro Tyr Thr 1 5 <210> 29 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR3 <400> 29 Gln His Ser Gly Glu Leu Pro Tyr 1 5 <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR3 <400> 30 Gln His Ser Gly Glu Leu Pro Tyr Thr 1 5 <210> 31 <211> 137 <212> PRT <213> Artificial Sequence <220> <223> Human TGF-betaRII <400> 31 Thr Ile Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val 1 5 10 15 Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys 20 25 30 Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn 35 40 45 Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala 50 55 60 Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His 65 70 75 80 Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser 85 90 95 Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe 100 105 110 Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser 115 120 125 Glu Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 32 <211> 198 <212> PRT <213> Artificial Sequence <220> <223> CD80 ECD <400> 32 Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys 1 5 10 15 Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp 20 25 30 Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn 35 40 45 Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn 50 55 60 Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr 65 70 75 80 Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His 85 90 95 Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser 100 105 110 Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys 115 120 125 Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn 130 135 140 Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu 145 150 155 160 Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr 165 170 175 Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn 180 185 190 Gln Thr Phe Asn Trp Asn 195 <210> 33 <211> 202 <212> PRT <213> Artificial Sequence <220> <223> VEGFR <400> 33 Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His 1 5 10 15 Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro 20 25 30 Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro 35 40 45 Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser 50 55 60 Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val 65 70 75 80 Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn 85 90 95 Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser 100 105 110 Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn 115 120 125 Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His 130 135 140 Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met 145 150 155 160 Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp 165 170 175 Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys 180 185 190 Asn Ser Thr Phe Val Arg Val His Glu Pro 195 200 <210> 34 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR1 <400> 34 Gly Ile Thr Phe Ser Asn Ser 1 5 <210> 35 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR1 <400> 35 Gly Ile Thr Phe Ser Asn Ser Gly Met His 1 5 10 <210> 36 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR1 <400> 36 Asn Ser Gly Met His 1 5 <210> 37 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR1 <400> 37 Ser Asn Ser Gly Met His 1 5 <210> 38 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR1 <400> 38 Gly Ile Thr Phe Ser Asn Ser Gly 1 5 <210> 39 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR2 <400> 39 Trp Tyr Asp Gly Ser Lys 1 5 <210> 40 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR2 <400> 40 Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr 1 5 10 <210> 41 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR2 <400> 41 Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 42 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR2 <400> 42 Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr 1 5 10 <210> 43 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR2 <400> 43 Ile Trp Tyr Asp Gly Ser Lys Arg 1 5 <210> 44 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR3 <400> 44 Asn Asp Asp Tyr One <210> 45 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR3 <400> 45 Asn Asp Asp Tyr One <210> 46 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR3 <400> 46 Asn Asp Asp Tyr One <210> 47 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR3 <400> 47 Ala Thr Asn Asp Asp 1 5 <210> 48 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR3 <400> 48 Ala Thr Asn Asp Asp Tyr 1 5 <210> 49 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR1 <400> 49 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 50 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR1 <400> 50 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 51 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR1 <400> 51 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 52 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR1 <400> 52 Ser Ser Tyr Leu Ala Trp Tyr 1 5 <210> 53 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR1 <400> 53 Gln Ser Val Ser Ser Tyr 1 5 <210> 54 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR2 <400> 54 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 55 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR2 <400> 55 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 56 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR2 <400> 56 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 57 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR2 <400> 57 Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala 1 5 10 <210> 58 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR2 <400> 58 Asp Ala Ser One <210> 59 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR3 <400> 59 Gln Gln Ser Ser Asn Trp Pro Arg Thr 1 5 <210> 60 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR3 <400> 60 Gln Gln Ser Ser Asn Trp Pro Arg Thr 1 5 <210> 61 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR3 <400> 61 Gln Gln Ser Ser Asn Trp Pro Arg Thr 1 5 <210> 62 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR3 <400> 62 Gln Gln Ser Ser Asn Trp Pro Arg 1 5 <210> 63 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region CDR3 <400> 63 Gln Gln Ser Ser Asn Trp Pro Arg Thr 1 5 <210> 64 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region amino acid sequence <400> 64 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <210> 65 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region amino acid sequence <400> 65 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 66 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region amino acid sequence <400> 66 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Arg Tyr 20 25 30 Ser Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Gly Val Gly Thr Thr Asp Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asn Trp Gly Thr Ala Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser 115 120 <210> 67 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region amino acid sequence <400> 67 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Lys Ser Val His Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr Tyr Cys Gln His Ser Gly 85 90 95 Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr <210> 68 <211> 668 <212> PRT <213> Artificial Sequence <220> <223> Hib-PDC heavy chain amino acid sequence <400> 68 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 180 185 190 Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Arg Val Glu Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 210 215 220 Gly Gly Gly Ser Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala 225 230 235 240 Thr Leu Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr 245 250 255 Arg Ile Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser 260 265 270 Gly Asp Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp 275 280 285 Ile Thr Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp 290 295 300 Glu Gly Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe 305 310 315 320 Lys Arg Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe 325 330 335 Pro Thr Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg 340 345 350 Arg Ile Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser 355 360 365 Trp Leu Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser 370 375 380 Gln Asp Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe 385 390 395 400 Asn Met Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His 405 410 415 Leu Arg Val Asn Gln Thr Phe Asn Trp Asn Gly Gly Gly Gly Ser Gly 420 425 430 Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Tyr Gly Pro Pro Cys Pro 435 440 445 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 450 455 460 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 465 470 475 480 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 485 490 495 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 500 505 510 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 515 520 525 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 530 535 540 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 545 550 555 560 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 565 570 575 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 580 585 590 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 595 600 605 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 610 615 620 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 625 630 635 640 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 645 650 655 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 660 665 <210> 69 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Hib-PDC light chain amino acid sequence <400> 69 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 70 <211> 672 <212> PRT <213> Artificial Sequence <220> <223> Hib-PDV heavy chain amino acid sequence <400> 70 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 180 185 190 Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Arg Val Glu Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 210 215 220 Gly Gly Gly Ser Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro 225 230 235 240 Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg 245 250 255 Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp 260 265 270 Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly 275 280 285 Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys 290 295 300 Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His 305 310 315 320 Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly 325 330 335 Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg 340 345 350 Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser 355 360 365 Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser 370 375 380 Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val 385 390 395 400 Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu 405 410 415 Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Pro Gly Gly 420 425 430 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Tyr Gly 435 440 445 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser 450 455 460 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 465 470 475 480 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 485 490 495 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 500 505 510 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 515 520 525 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 530 535 540 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 545 550 555 560 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 565 570 575 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 580 585 590 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 595 600 605 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 610 615 620 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 625 630 635 640 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 645 650 655 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 660 665 670 <210> 71 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Hib-PDV light chain amino acid sequence <400> 71 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 72 <211> 615 <212> PRT <213> Artificial Sequence <220> <223> Hib-PLT heavy chain amino acid sequence <400> 72 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Arg Tyr 20 25 30 Ser Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Gly Val Gly Thr Thr Asp Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asn Trp Gly Thr Ala Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Gly 210 215 220 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Thr Ile 225 230 235 240 Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp 245 250 255 Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val 260 265 270 Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser 275 280 285 Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp 290 295 300 Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro 305 310 315 320 Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys 325 330 335 Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys 340 345 350 Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu 355 360 365 Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly 370 375 380 Ser Gly Gly Gly Gly Ser Thr His Thr Cys Pro Pro Cys Pro Ala Pro 385 390 395 400 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Lys Pro Lys 405 410 415 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 420 425 430 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 435 440 445 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 450 455 460 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 465 470 475 480 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 485 490 495 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 500 505 510 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 515 520 525 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 530 535 540 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 545 550 555 560 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 565 570 575 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 580 585 590 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 595 600 605 Leu Ser Leu Ser Pro Gly Lys 610 615 <210> 73 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> Hib-PLT light chain amino acid sequence <400> 73 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Lys Ser Val His Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr Tyr Cys Gln His Ser Gly 85 90 95 Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 74 <211> 2007 <212> DNA <213> Artificial Sequence <220> <223> Hib-PDC heavy chain nucleic acid sequence <400> 74 caggtgcagc tcgtggagag cgggggaggc gtcgtccagc ctggccgtag cctgcggctg 60 gactgtaagg ctagcggaat cacgttcagc aacagcggaa tgcactgggt cagacaggct 120 cctggcaaag gcctcgaatg ggtcgccgtg atctggtacg acgggagcaa gagatactac 180 gcagattcag tgaagggccg tttcacaatc agccgtgaca actcgaagaa cacactgttc 240 ctgcagatga acagcctgag ggcagaagac acagcagtct actactgcgc tacaaatgac 300 gactactggg gccagggaac actggtcacc gtgagctcag cttccaccaa gggcccatcc 360 gtcttccccc tggcgccctg ctccaggagc acctccgaga gcacagccgc cctgggctgc 420 ctggtcaagg actacttccc cgaaccggtg acggtgtcgt ggaactcagg cgccctgacc 480 agcggcgtgc acaccttccc ggctgtccta cagtcctcag gactctactc cctcagcagc 540 gtggtgaccg tgccctccag cagcttgggc acgaagacct acacctgcaa cgtagatcac 600 aagcccagca acaccaaggt ggacaagaga gttgagtccg gtggcggcgg aagcggcggc 660 ggaggcagcg gcggaggcgg cagcgtgatc catgtcacga aggaggtcaa agaagtggcc 720 accctcagct gcggtcacaa cgtcagcgtg gaagagttgg cccagaccag aatctactgg 780 cagaaggaga agaagatggt cttgaccatg atgagcggcg acatgaacat ctggccagag 840 tacaagaata gaactatctt cgacatcacc aataacctga gcatcgtgat cctcgctttg 900 cgtccgagcg acgaaggcac ctacgagtgc gtagtgctaa agtacgagaa agacgccttc 960 aagcgggagc acctggcaga agtaaccctg agcgtgaagg cagacttccc aacgcctagc 1020 atctccgact tcgagatccc cacaagcaac atccggcgga tcatctgtag cacctccggg 1080 ggtttccccg agcctcacct gtcctggctc gagaacggcg aggagctgaa cgccatcaac 1140 accaccgtga gccaggaccc cgagacagaa ctgtacgccg tgagctccaa gctcgacttc 1200 aacatgacca caaatcacag cttcatgtgc ctcatcaagt acggacacct gcgggtgaat 1260 cagacgttca actggaacgg cggggggggt agcggcggcg ggggctcagg tggggggggc 1320 tcaaaatatg gtcccccatg cccaccatgc ccagcacctg agttcctggg gggaccatca 1380 gtcttcctgt tccccccaaa acccaaggac actctcatga tctcccggac ccctgaggtc 1440 acgtgcgtgg tggtggacgt gagccaggaa gaccccgagg tccagttcaa ctggtacgtg 1500 gatggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagtt caacagcacg 1560 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaacgg caaggagtac 1620 aagtgcaagg tctccaacaa aggcctcccg tcctccatcg agaaaaccat ctccaaagcc 1680 aaagggcagc cccgagagcc acaggtgtac accctgcccc catcccagga ggagatgacc 1740 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct accccagcga catcgccgtg 1800 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1860 tccgacggct ccttcttcct ctacagcagg ctaaccgtgg acaagagcag gtggcaggag 1920 gggaatgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacacagaag 1980 agcctctccc tgtctctggg taaatag 2007 <210> 75 <211> 645 <212> DNA <213> Artificial Sequence <220> <223> Hib-PDC light chain nucleic acid sequence <400> 75 gagatcgtgc tgacacagag cccagccact ctgtcactgt ccccaggaga aagggctact 60 ctgtcttgcc gggcaagcca gtctgtctcc agctacctgg cctggtatca gcagaagccc 120 ggacaggctc ctagactgct gatctacgac gcaagtaaca gagccaccgg catccccgca 180 cgcttcagtg gctcaggctc cggaacagac tttactctga ccatctctag tctggagcct 240 gaagatttcg ccgtgtacta ttgtcagcag agctctaatt ggcctagaac cttcggccag 300 ggcaccaaag tcgagatcaa gcgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 645 <210> 76 <211> 2019 <212> DNA <213> Artificial Sequence <220> <223> Hib-PDV heavy chain nucleic acid sequence <400> 76 caggtgcagc tcgtggagag cgggggaggc gtcgtccagc ctggccgtag cctgcggctg 60 gactgtaagg ctagcggaat cacgttcagc aacagcggaa tgcactgggt cagacaggct 120 cctggcaaag gcctcgaatg ggtcgccgtg atctggtacg acgggagcaa gagatactac 180 gcagattcag tgaagggccg tttcacaatc agccgtgaca actcgaagaa cacactgttc 240 ctgcagatga acagcctgag ggcagaagac acagcagtct actactgcgc tacaaatgac 300 gactactggg gccagggaac actggtcacc gtgagctcag cttccaccaa gggcccatcc 360 gtcttccccc tggcgccctg ctccaggagc acctccgaga gcacagccgc cctgggctgc 420 ctggtcaagg actacttccc cgaaccggtg acggtgtcgt ggaactcagg cgccctgacc 480 agcggcgtgc acaccttccc ggctgtccta cagtcctcag gactctactc cctcagcagc 540 gtggtgaccg tgccctccag cagcttgggc acgaagacct acacctgcaa cgtagatcac 600 aagcccagca acaccaaggt ggacaagaga gttgagtccg gtggcggcgg aagcggcggc 660 ggaggcagcg gcggaggcgg cagcggtaga ccattcgtag agatgtacag cgaaatcccc 720 gaaatcatcc acatgactga aggaagggag ctcgtcatcc cctgccgggt tacgtcacct 780 aacatcactg ttactctgaa gaagttccca ctcgacactt tgatccctga tggaaaacgc 840 atcatctggg acagcagaaa gggcttcatc atctcaaatg caacgtacaa agaaatcgga 900 ctcctgacct gtgaagcaac agtcaatgga catttgtata agacaaacta tctcacacat 960 cgacagacca atacaatcat cgatgtggtt ctgagcccgt ctcatggaat cgaactatct 1020 gttggagaaa agctcgtcct gaattgtaca gcaagaactg aactgaatgt gggaatcgac 1080 ttcaactggg aatacccttc ttcgaagcat cagcataaga aactcgtaaa ccgagaccta 1140 aagacccagt ctgggagcga gatgaagaaa ttcttgagca ccctgactat cgatggtgta 1200 acccggagcg accagggatt gtacacctgt gcagcatcca gcgggctgat gaccaagaag 1260 aacagcacat tcgtcagggt ccatgaaccc ggcgggggg gtagcggcgg cgggggctca 1320 ggtggggggg gctcaaaata tggtccccca tgcccaccat gcccagcacc tgagttcctg 1380 gggggaccat cagtcttcct gttcccccca aaacccaagg acactctcat gatctcccgg 1440 acccctgagg tcacgtgcgt ggtggtggac gtgagccagg aagaccccga ggtccagttc 1500 aactggtacg tggatggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 1560 ttcaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaac 1620 ggcaaggagt acaagtgcaa ggtctccaac aaaggcctcc cgtcctccat cgagaaaacc 1680 atctccaaag ccaaagggca gccccgagag ccacaggtgt acaccctgcc cccatcccag 1740 gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctaccccagc 1800 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1860 cccgtgctgg actccgacgg ctccttcttc ctctacagca ggctaaccgt ggacaagagc 1920 aggtggcagg aggggaatgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1980 tacacacaga agagcctctc cctgtctctg ggtaaatag 2019 <210> 77 <211> 645 <212> DNA <213> Artificial Sequence <220> <223> Hib-PDV light chain nucleic acid sequence <400> 77 gagatcgtgc tgacacagag cccagccact ctgtcactgt ccccaggaga aagggctact 60 ctgtcttgcc gggcaagcca gtctgtctcc agctacctgg cctggtatca gcagaagccc 120 ggacaggctc ctagactgct gatctacgac gcaagtaaca gagccaccgg catccccgca 180 cgcttcagtg gctcaggctc cggaacagac tttactctga ccatctctag tctggagcct 240 gaagatttcg ccgtgtacta ttgtcagcag agctctaatt ggcctagaac cttcggccag 300 ggcaccaaag tcgagatcaa gcgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 645 <210> 78 <211> 1848 <212> DNA <213> Artificial Sequence <220> <223> Hib-PLT light chain nucleic acid sequence <400> 78 caggtgcagc tgcaggagtc cggaccagga ctggtgaagc catccgagac cctgagcctg 60 acctgtacag tgtccggctt cagcctgtct aggtacagcg tgcactggat cagacagcca 120 cctggcaagg gactggagtg gctgggcatg atctggggcg tgggcaccac agactacaac 180 tctgctctga agtccagact gaccatcagc aaggatacat ctaagaatca gttcagcctg 240 aagctgtcca gcgtgaccgc cgctgacaca gccgtgtact attgcgctcg caactggggc 300 accgccgact acttcgacta ttggggccag ggcaccacag tgacagtgtc ttccgctagc 360 accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tggggggcaca 420 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540 tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600 tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 660 tgtgacaaag gcgggggagg cagcggcggc ggaggaagcg ggggcggagg tagcaccatc 720 cctccacacg tgcagaagag cgtgaacaat gacatgatcg tcaccgacaa caacggcgcc 780 gtcaagttcc cccagctgtg caaattctgc gacgtgaggt tctccacgtg cgacaaccag 840 aagagctgta tgagcaactg cagcatcaca tccatctgcg aaaaacccca ggaagtgtgc 900 gtcgccgtct ggcggaagaa cgacgagaac atcacactgg agaccgtgtg ccacgacccc 960 aaactgccct accacgactt catcctggag gacgccgcca gcccaaagtg catcatgaaa 1020 gagaagaaga agccgggcga gactttcttc atgtgctcct gcagctccga cgagtgcaac 1080 gataatatca tcttcagcga agaatacaac acatctaacc cagacggagg gggcggatcc 1140 gggggcggcg gaagcggcgg ggggggcagc actcacacat gcccaccgtg cccagcacct 1200 gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 1260 atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 1320 gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 1380 gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1440 tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1500 gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 1560 ccatccccggg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1620 tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1680 accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1740 gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1800 cacaaccact acaccgcagaa gagcctctcc ctgtctccgg gtaaatga 1848 <210> 79 <211> 657 <212> DNA <213> Artificial Sequence <220> <223> Hib-PLT light chain nucleic acid sequence <400> 79 gatatcgtgc tgacccagtc tccagcttcc ctggccgtgt ccccaggaca gagggccacc 60 atcacatgtc gggcttccaa gagcgtgcac acaagcggct actcttatat gcattggtac 120 cagcagaagc ccggccagcc ccctaagctg ctgatctatc tggcttccaa cctggagagc 180 ggagtgccag ctaggttctc tggctccggc agcggcaccg actttaccct gacaatcaat 240 cctgtggagg ccaacgatac agctaattac tattgccagc actccggaga gctgccatac 300 accttcggcg gaggcacaaa ggtggagatc aagcgtacgg tggctgcacc atctgtcttc 360 atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420 aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg 480 ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc 540 agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc 600 acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgttag 657 <210> 80 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 80 Ala Lys Thr Thr Pro Lys Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg 1 5 10 15 <210> 81 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 81 Ala Lys Thr Thr Pro Lys Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg 1 5 10 15 Val <210> 82 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 82 Ala Lys Thr Thr Pro Lys Leu Gly Gly 1 5 <210> 83 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 83 Ser Ala Lys Thr Thr Pro Lys Leu Gly Gly 1 5 10 <210> 84 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 84 Ser Ala Lys Thr Thr Pro 1 5 <210> 85 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 85 Arg Ala Asp Ala Ala Pro 1 5 <210> 86 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 86 Arg Ala Asp Ala Ala Pro Thr Val Ser 1 5 <210> 87 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 87 Arg Ala Asp Ala Ala Ala Ala Gly Gly Pro Gly Ser 1 5 10 <210> 88 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 88 Arg Ala Asp Ala Ala Ala Ala 1 5 <210> 89 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 89 Ser Ala Lys Thr Thr Pro Lys Leu Glu Glu Gly Glu Phe Ser Glu Ala 1 5 10 15 Arg Val <210> 90 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 90 Ala Asp Ala Ala Pro 1 5 <210> 91 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 91 Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro 1 5 10 <210> 92 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 92 Thr Val Ala Ala Pro 1 5 <210> 93 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 93 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 1 5 10 <210> 94 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 94 Gln Pro Lys Ala Ala Pro 1 5 <210> 95 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 95 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro 1 5 10 <210> 96 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 96 Ala Lys Thr Thr Pro Pro 1 5 <210> 97 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 97 Ala Lys Thr Thr Pro Ser Val Thr Pro Leu Ala Pro 1 5 10 <210> 98 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 98 Ala Lys Thr Thr Ala Pro 1 5 <210> 99 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 99 Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro 1 5 10 <210> 100 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 100 Ala Ser Thr Lys Gly Pro 1 5 <210> 101 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 101 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 1 5 10 <210> 102 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 102 Gly Glu Asn Lys Val Glu Tyr Ala Pro Ala Leu Met Ala Leu Ser 1 5 10 15 <210> 103 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 103 Gly Pro Ala Lys Glu Leu Thr Pro Leu Lys Glu Ala Lys Val Ser 1 5 10 15 <210> 104 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 104 Gly His Glu Ala Ala Ala Val Met Gln Val Gln Tyr Pro Ala Ser 1 5 10 15 <210> 105 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 105 Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala 1 5 10 15 <210> 106 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain amino acid sequence of Atezolizumab <400> 106 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 107 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Light chain amino acid sequence of Atezolizumab <400> 107 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 108 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain amino acid sequence of Avelumab <400> 108 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 109 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Light chain amino acid sequence of Avelumab <400> 109 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215 <210> 110 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain amino acid sequence of Durvalumab <400> 110 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Gly Trp Phe Gly Glu Leu Ala Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 111 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Light chain amino acid sequence of Durvalumab <400> 111 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Arg Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Asp Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Leu Pro 85 90 95 Trp Thr Phe Gly Gin Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 112 <211> 440 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain amino acid sequence of Nivolumab <400> 112 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 180 185 190 Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 210 215 220 Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 225 230 235 240 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 245 250 255 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 260 265 270 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 275 280 285 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 290 295 300 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 305 310 315 320 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 325 330 335 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 340 345 350 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 355 360 365 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 370 375 380 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 385 390 395 400 Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 405 410 415 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 420 425 430 Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 113 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Light chain amino acid sequence of Nivolumab <400> 113 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 114 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Pembrolizumab heavy chain amino acid sequence <400> 114 Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 115 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> Pembrolizumab light chain amino acid sequence <400> 115 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 116 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> Ipilimumab heavy chain amino acid sequence <400> 116 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 117 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Ipilimumab light chain amino acid sequence <400> 117 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gin Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 118 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> Cemiplimab heavy chain amino acid sequence <400> 118 Glu Val Gln Leu Leu Glu Ser Gly Gly Val Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe 20 25 30 Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Gly Gly Gly Arg Asp Thr Tyr Phe Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Gly Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Lys Trp Gly Asn Ile Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 119 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Cemiplimab light chain amino acid sequence <400> 119 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Ser Ile Thr Ile Thr Cys Arg Ala Ser Leu Ser Ile Asn Thr Phe 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu His Gly Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Thr Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ser Asn Thr Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Val Val Asp Phe Arg Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 120 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 120 Gly Gly Gly Gly Ser 1 5 <210> 121 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 121 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 122 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 122 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 123 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 123 Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 124 <211> 600 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain amino acid sequence of Control protein 1 <400> 124 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Arg Tyr 20 25 30 Ser Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Gly Val Gly Thr Thr Asp Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asn Trp Gly Thr Ala Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr 450 455 460 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 465 470 475 480 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 485 490 495 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 500 505 510 Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 515 520 525 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 530 535 540 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 545 550 555 560 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 565 570 575 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 580 585 590 Glu Tyr Asn Thr Ser Asn Pro Asp 595 600 <210> 125 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> Light chain amino acid sequence of Control protein 1 <400> 125 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Lys Ser Val His Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr Tyr Cys Gln His Ser Gly 85 90 95 Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 126 <211> 653 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain amino acid sequence of Control protein 2 <400> 126 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 180 185 190 Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 210 215 220 Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 225 230 235 240 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 245 250 255 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 260 265 270 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 275 280 285 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 290 295 300 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 305 310 315 320 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 325 330 335 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 340 345 350 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 355 360 365 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 370 375 380 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 385 390 395 400 Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 405 410 415 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 420 425 430 Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly 435 440 445 Gly Ser Gly Gly Gly Gly Ser Val Ile His Val Thr Lys Glu Val Lys 450 455 460 Glu Val Ala Thr Leu Ser Cys Gly His Asn Val Ser Val Glu Glu Leu 465 470 475 480 Ala Gln Thr Arg Ile Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr 485 490 495 Met Met Ser Gly Asp Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr 500 505 510 Ile Phe Asp Ile Thr Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg 515 520 525 Pro Ser Asp Glu Gly Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys 530 535 540 Asp Ala Phe Lys Arg Glu His Leu Ala Glu Val Thr Leu Ser Val Lys 545 550 555 560 Ala Asp Phe Pro Thr Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser 565 570 575 Asn Ile Arg Arg Ile Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro 580 585 590 His Leu Ser Trp Leu Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr 595 600 605 Thr Val Ser Gln Asp Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys 610 615 620 Leu Asp Phe Asn Met Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys 625 630 635 640 Tyr Gly His Leu Arg Val Asn Gln Thr Phe Asn Trp Asn 645 650 <210> 127 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Light chain amino acid sequence of Control protein 2 <400> 127 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 128 <211> 657 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain amino acid sequence of Control protein 3 <400> 128 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 180 185 190 Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 210 215 220 Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 225 230 235 240 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 245 250 255 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 260 265 270 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 275 280 285 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 290 295 300 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 305 310 315 320 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 325 330 335 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 340 345 350 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 355 360 365 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 370 375 380 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 385 390 395 400 Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 405 410 415 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 420 425 430 Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly 435 440 445 Gly Ser Gly Gly Gly Gly Ser Gly Arg Pro Phe Val Glu Met Tyr Ser 450 455 460 Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile 465 470 475 480 Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe 485 490 495 Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser 500 505 510 Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu 515 520 525 Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr 530 535 540 Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro 545 550 555 560 Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys 565 570 575 Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr 580 585 590 Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys 595 600 605 Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile 610 615 620 Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser 625 630 635 640 Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu 645 650 655 Pro <210> 129 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Light chain amino acid sequence of Control protein 3 <400> 129 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 130 <211> 202 <212> PRT <213> Artificial Sequence <220> <223> VEGF-R fragment <400> 130 Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His 1 5 10 15 Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro 20 25 30 Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro 35 40 45 Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser 50 55 60 Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val 65 70 75 80 Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn 85 90 95 Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser 100 105 110 Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn 115 120 125 Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His 130 135 140 Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met 145 150 155 160 Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp 165 170 175 Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys 180 185 190 Asn Ser Thr Phe Val Arg Val His Glu Pro 195 200 <210> 131 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Human TGF-betaRII <400> 131 Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe 1 5 10 15 Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr 20 25 30 Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys 35 40 45 Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu 50 55 60 Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile 65 70 75 80 Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys 85 90 95 Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn 100 105 110 Thr Ser Asn Pro Asp 115 <210> 132 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Human TGF-betaRII <400> 132 Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr 1 5 10 15 Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile 20 25 30 Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp 35 40 45 Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr 50 55 60 His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys 65 70 75 80 Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser 85 90 95 Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser 100 105 110 Asn Pro Asp 115 <210> 133 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> CD80 ECD <400> 133 Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys 1 5 10 15 Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp 20 25 30 Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn 35 40 45 Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn 50 55 60 Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr 65 70 75 80 Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His 85 90 95 Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser 100 105 110 <210> 134 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> CD80 <400> 134 Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys Cys Pro Tyr 1 5 10 15 Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys 20 25 30 Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu 35 40 45 Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile 50 55 60 Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp 65 70 75 80 Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr 85 90 95 Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly 100 105 110 Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg 115 120 125 Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr 130 135 140 Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile 145 150 155 160 Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu 165 170 175 Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp 180 185 190 Pro Glu Thr Glu Leu Tyr Ala Val Ser Lys Leu Asp Phe Asn Met 195 200 205 Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg 210 215 220 Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro 225 230 235 240 Asp Asn Leu Leu Pro Ser Trp Ala Ile Thr Leu Ile Ser Val Asn Gly 245 250 255 Ile Phe Val Ile Cys Cys Leu Thr Tyr Cys Phe Ala Pro Arg Cys Arg 260 265 270 Glu Arg Arg Arg Asn Glu Arg Leu Arg Arg Glu Ser Val Arg Pro Val 275 280 285 <210> 135 <211> 256 <212> PRT <213> Artificial Sequence <220> <223> CD80 <400> 135 Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys Cys Pro Tyr 1 5 10 15 Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys 20 25 30 Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu 35 40 45 Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile 50 55 60 Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp 65 70 75 80 Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr 85 90 95 Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly 100 105 110 Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg 115 120 125 Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr 130 135 140 Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile 145 150 155 160 Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu 165 170 175 Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp 180 185 190 Pro Glu Thr Glu Leu Tyr Ala Val Ser Lys Leu Asp Phe Asn Met 195 200 205 Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg 210 215 220 Val Asn Gln Thr Phe Asn Trp Asn Thr Ser Phe Ala Pro Arg Cys Arg 225 230 235 240 Glu Arg Arg Arg Asn Glu Arg Leu Arg Arg Glu Ser Val Arg Pro Val 245 250 255 <210> 136 <211> 162 <212> PRT <213> Artificial Sequence <220> <223> CD80 <400> 136 Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys Cys Pro Tyr 1 5 10 15 Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys 20 25 30 Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu 35 40 45 Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile 50 55 60 Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp 65 70 75 80 Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr 85 90 95 Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly 100 105 110 Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg 115 120 125 Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Gly Phe Ala Pro Arg 130 135 140 Cys Arg Glu Arg Arg Arg Asn Glu Arg Leu Arg Arg Glu Ser Val Arg 145 150 155 160 Pro Val <210> 137 <211> 137 <212> PRT <213> Artificial Sequence <220> <223> TGF-betaRII extracellular region fragment <400> 137 Thr Ile Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val 1 5 10 15 Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys 20 25 30 Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn 35 40 45 Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala 50 55 60 Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His 65 70 75 80 Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser 85 90 95 Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe 100 105 110 Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser 115 120 125 Glu Glu Tyr Asn Thr Ser Asn Pro Asp 130 135

Claims (45)

A bispecific fusion protein comprising:
the fusion protein has a structure in which a second binding domain is inserted through an optional peptide linker into the hinge region of a full-length immunoglobulin G (IgG), wherein the Fab region of the IgG is a first binding domain;
after the second binding domain is inserted, the heavy chain of the IgG is the heavy chain of the fusion protein, and the light chain of the IgG is the light chain of the fusion protein;
wherein said second binding domain is selected from a combination of a human receptor or ligand, a fragment of said receptor or ligand, and a fragment of said receptor or ligand;
wherein the receptor or ligand forms a dimerization or multimerization structure in a natural signaling pathway to activate or inhibit a signaling pathway, and wherein the second binding domain and the first binding domain target different targets. protein.
The fusion protein of claim 1 , wherein the first binding domain targets and binds an immune checkpoint molecule or a tumor antigen.
3. The method of claim 2, wherein the immune checkpoint molecule is selected from: PD-1, PD-L1, CTLA-4, LAG-3, FGL1, TIM-3, galectin-9, TIGIT, CD155 and CD47; and claudin 18.2, HER-2, mesothelin, BCMA, SSTR2, GPRC5D, PSMA, FCRH5, CD33, CD123, CD20, A33, CEA, CD28, DLL3, EGFR, VEGFR, VEGFR2, VEGF-A, Nectin-4, FGFR , a fusion protein comprising a tumor antigen comprising C-met, RANKL, PDGF, PDGFR, PDGFRα, DLL4, Ang-1 and Ang-2.
4. The method of claim 3, wherein the second binding domain is human TGF-β, CTLA-4, VEGF, LAG3, CD27, 4-1BB, OX40, CD47, FGL1, TLT-2, CD28, HGF, CSF1, CXCL1, CXCL2 , CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL12, GITR, EGF and ICOSL by targeting and binding.
5. The method of claim 4, wherein the receptor or ligand is human TGF-β receptor (TGF-βR), CD80, CD86, VEGFR, VEGF-trap, FGL1, CD70, 4-1BBL, OX40L, SIRPα, B7-H3 , C- met, CSF1R, CXCR2, CXCR3, CXCR4, GITRL, EGFR and ICOS.
5. The fusion protein of claim 4, wherein the first binding domain and the second binding domain are selected from the following combinations:
(1) the first binding domain targets and binds to PD-L1, and the second binding domain is human TGF-β, VEGF, FGL1, CD47, CD155, HGF, CSF1, CXCL1, CXCL2, CXCL3, CXCL5, target and bind CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL12, EGF or ICOSL; or
(2) the first binding domain targets and binds to PD-1, and the second binding domain targets and targets human CTLA-4, VEGF, HGF, EGF, CD28, LAG3, CD27, 4-1BB or OX40. to combine; or
(3) the first binding domain targets and binds CTLA-4, and the second binding domain targets and binds human CD28, VEGF, HGF, EGF, LAG3, CD27, 4-1BB or OX40; or
(4) the first binding domain targets and binds to LAG-3, and the second binding domain targets and binds to human CD28, VEGF, HGF, EGF, CTLA-4, CD27, 4-1BB or OX40 do or; or
(5) the first binding domain targets and binds to FGL1, and the second binding domain is human TGF-β, VEGF, CD47, CD155, HGF, CSF1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, target and bind CXCL8, CXCL9, CXCL10, CXCL12, EGF or ICOSL; or
(6) the first binding domain targets and binds TIM-3, and the second binding domain targets and binds human VEGF, HGF, EGF, LAG3, CD27, 4-1BB or OX40; or
(7) the first binding domain targets and binds to galectin-9, and the second binding domain is human TGF-β, VEGF, CD47, CD155, HGF, CSF1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6 , CXCL7, CXCL8, CXCL9, CXCL10, CXCL12, EGF or ICOSL by targeting; or
(8) the first binding domain targets and binds TIGIT, and the second binding domain targets and binds human TGF-β, HGF, EGF or VEGF; or
(9) the first binding domain targets and binds CD155, and the second binding domain targets and binds human TGF-β, VEGF, HGF, EGF, CD47 or CD155; or
(10) the first binding domain is claudin 18.2, HER-2, mesothelin, BCMA, SSTR2, GPRC5D, PSMA, FCRH5, CD33, CD123, CD20, A33, CEA, CD28, DLL3, EGFR, VEGFR, VEGFR2, VEGF -A, nectin-4, FGFR, C-met, RANKL, PDGF, PDGFR, PDGFRα, DLL-4, Ang-1 or Ang-2 target and bind, wherein the second binding domain is human CTLA-4, TGF-β, VEGF, FGL1, LAG3, 4-1BB, OX40, CD27, CD28, CD47, CD155, HGF, CSF1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL12, EGF or Bind by targeting ICOSL.
7. The fusion protein of claim 6, wherein said first binding domain and said receptor or ligand are selected from the following combinations:
(1) the first binding domain binds to target PD-L1, and the receptor or ligand is TGF-βRII, VEGFR, LAG-3, SIRPα, TIGIT, C-MET, CSF1R, CXCR2, CXCR3, CXCR4, EGFR or ICOS; or
(2) the first binding domain targets and binds PD-1, and the receptor or ligand is selected from human CD80, CD86, VEGFR, c-MET, EGFR, FGL1, CD70, 4-1BBL or OX40L; or
(3) the first binding domain targets and binds CTLA-4, and the receptor or ligand is selected from human CD80, CD86, VEGFR, c-MET, EGFR, FGL1, CD70, 4-1BBL or OX40L; or
(4) the first binding domain targets and binds LAG-3, and the receptor or ligand is selected from human VEGFR, c-MET, EGFR, CD80, CD86, CD70, 4-1BBL or OX40L; or
(5) the first binding domain binds by targeting FGL1, and the receptor or ligand is selected from human TGF-βRII, VEGFR, SIRPα, TIGIT, c-MET, CSF1R, CXCR2, CXCR3, CXCR4, EGFR or ICOS or; or
(6) the first binding domain binds by targeting TIM-3, and the receptor or ligand is selected from human VEGFR, c-MET, EGFR, FGL1, CD70, 4-1BBL or OX40L; or
(7) the first binding domain targets and binds to galectin-9, and the receptor or ligand is human TGF-βRII, VEGFR, SIRPα, TIGIT, c-MET, CSF1R, CXCR2, CXCR3, CXCR4, EGFR or selected from ICOS; or
(8) the first binding domain binds by targeting TIGIT, and the receptor or ligand is selected from human TGF-βRII, c-MET, EGFR or VEGFR; or
(9) the first binding domain targets and binds CD155, and the receptor or ligand is selected from human TGF-βRII, VEGFR, c-MET, EGFR, SIRPα or TIGIT; or
(10) the first binding domain is claudin 18.2, HER-2, mesothelin, BCMA, SSTR2, GPRC5D, PSMA, FCRH5, CD33, CD123, CD20, A33, CEA, CD28, DLL3, EGFR, VEGFR, VEGFR2, Nectin Targets and binds to -4, FGFR, c-MET, RANKL, PDGF, PDGFR, PDGFRα, DLL4, Ang-1 or Ang-2, wherein the receptor or ligand is human CTLA-4, CD80, CD86, TGF-βRII , VEGFR, FGL1, LAG3, 4-1BB, OX40, CD27, CD28, CD70, c-MET, SIRPα, TIGIT, CSF1R, CXCR2, CXCR3, CXCR4, EGFR or ICOS.
8. The fusion protein according to any one of claims 1 to 7, wherein the fragment of the receptor or ligand comprises a fragment of an extracellular region and a fragment of the binding domain of the receptor or ligand.
8. The method of claim 7, wherein the first binding domain targets and binds PD-L1; said second binding domain is a fragment of human TGF-βRII; or the first binding domain binds by targeting PD-1, and the second binding domain comprises a second extracellular region of human CD80 ECD, CD80 IgV region, human CD80 IgVIgC, VEGFR1 ECD, VEGFR2 ECD or VEGFR1 and VEGFR2 selected from a combination of the third extracellular region of; or the first binding domain binds by targeting claudin 18.2, HER-2 or EGFR, and the second binding domain is a human CD80 ECD, CD80 IgV region, human CD80 IgVIgC, VEGFR1 ECD, VEGFR2 ECD or VEGFR1 second A fusion protein, which is selected from a combination of an extracellular region and a third extracellular region of VEGFR2.
10. The fusion protein of claim 9, wherein said second binding domain is selected from:
(1) 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 compared to the sequence shown in SEQ ID NO: 31, 131 or 132, or the sequence shown in SEQ ID NO: 31, 131 or 132 % homology, or an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions or deletions on the sequence shown in SEQ ID NO: 31, 131 or 132 , human TGF-βRII; or
(2) at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology to the sequence shown in SEQ ID NO: 32 or 133, or to the sequence shown in SEQ ID NO: 32 or 133 a CD80 ECD comprising an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions or deletions in the sequence shown in SEQ ID NO: 32 or 133; or
(3) a combination of the second extracellular region of VEGFR1 and the third extracellular region of VEGFR2, wherein the sequence shown in SEQ ID NO: 33, or 80%, 85%, 90%, 95% compared to the sequence shown in SEQ ID NO: 33 , 96%, 97%, 98%, 99% or more homology, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions on the sequence shown in SEQ ID NO: 33 or combinations comprising deletions.
The method according to claim 9 or 10, wherein the first binding domain targets and binds to PD-L1, and the CDRs of the heavy chain variable region and/or the CDRs of the light chain variable region in the Fab of the IgG have the following sequence A fusion protein having the same CDR sequence as the antibody as defined, or having 1-2 amino acid substitutions in the CDR of the antibody as defined by the following sequence, wherein the antibody is defined as follows:
(1) the amino acid sequence of the heavy chain variable region of SEQ ID NO: 66; and/or
(2) the amino acid sequence of the light chain variable region of SEQ ID NO: 67.
The fusion protein according to claim 11, wherein the CDRs of the heavy chain variable region and/or the CDRs of the light chain variable region in the Fab of the IgG are as follows:
(1) for the heavy chain variable region, the amino acid sequence of CDR1 is selected from amino acid sequences having 1 or 2 amino acid substitutions in SEQ ID NO: 1-5 and SEQ ID NO: 1-5; the amino acid sequence of CDR2 is selected from amino acid sequences having 1 or 2 amino acid substitutions in SEQ ID NO: 6-10 and SEQ ID NO: 6-10; the amino acid sequence of CDR3 is selected from the amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 11-15 and SEQ ID NO: 11-15; and/or
(2) for the light chain variable region, the amino acid sequence of CDR1 is selected from amino acid sequences having 1 or 2 amino acid substitutions in SEQ ID NOs: 16-20 and SEQ ID NOs: 16-20; the amino acid sequence of CDR2 is selected from the amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 21-25 and SEQ ID NO: 21-25; The amino acid sequence of CDR3 is selected from amino acid sequences having 1 or 2 amino acid substitutions in SEQ ID NOs: 26-30 and SEQ ID NOs: 26-30.
13. The method of claim 12,
(1) the amino acid sequence of CDRs 1-3 of the heavy chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 1, 6, 11, or SEQ ID NO: 1, 6, 11; and/or the amino acid sequence of CDRs 1-3 of the light chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 16, 21, 26, or SEQ ID NO: 16, 21, 26; or
(2) the amino acid sequence of CDRs 1-3 of the heavy chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 2, 7, 12, or SEQ ID NO: 2, 7, 12; and/or the amino acid sequence of CDRs 1-3 of the light chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 17, 22, 27, or SEQ ID NO: 17, 22, 27; or
(3) the amino acid sequence of CDRs 1-3 of the heavy chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 3, 8, 13 or SEQ ID NO: 3, 8, 13; and/or the amino acid sequence of CDRs 1-3 of the light chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 18, 23, 28, or SEQ ID NO: 18, 23, 28; or
(4) the amino acid sequence of CDRs 1-3 of the heavy chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 4, 9, 14, or SEQ ID NO: 4, 9, 14; and/or the amino acid sequence of CDRs 1-3 of the light chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 19, 24, 29, or SEQ ID NO: 19, 24, 29; or
(5) the amino acid sequence of CDRs 1-3 of the heavy chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 5, 10, 15 or SEQ ID NO: 5, 10, 15; and/or the amino acid sequence of CDRs 1-3 of the light chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 20, 25, 30, or SEQ ID NO: 20, 25, 30.
The method of claim 13, wherein the amino acid sequence of CDRs 1-3 of the heavy chain variable region is SEQ ID NO: 3, 8, 13; And/or the amino acid sequence of CDRs 1-3 of the light chain variable region is SEQ ID NO: 18, 23, 28, the fusion protein.
15. The method of claim 14,
(1) the heavy chain variable region has the sequence shown in SEQ ID NO: 66, or CDRs 1-3 identical to SEQ ID NO: 66, and has 80%, 85%, 90%, 95%, 96 homology compared to SEQ ID NO: 66 %, 97%, 98%, 99% or more of the sequence; and/or
(2) the light chain variable region has the sequence shown in SEQ ID NO: 67, or CDRs 1-3 identical to that of SEQ ID NO: 67, and has 80%, 85%, 90%, 95%, 96% homology compared to SEQ ID NO: 67 , 97%, 98%, 99% or more of the sequence.
16. The method of claim 15,
(1) the heavy chain of the fusion protein has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence shown in SEQ ID NO: 72, or SEQ ID NO: 72 has a sequence with;
(2) the light chain of the fusion protein has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology compared to the amino acid sequence shown in SEQ ID NO: 73, or SEQ ID NO: 73 , which has a sequence of more than one, the fusion protein.
17. The method of claim 16,
(1) the amino acid sequence of the heavy chain of the fusion protein has 1-15 amino acid site mutations compared to SEQ ID NO: 72 or has an alternative peptide linker;
(2) the amino acid sequence of the light chain of the fusion protein has 1-10 amino acid site mutations compared to SEQ ID NO: 73, the fusion protein.
10. The method of claim 9,
The first binding domain binds to target PD-1, and the CDRs of the heavy chain variable region and/or the CDRs of the light chain variable region in the Fab of IgG have the same CDR sequence as the antibody defined by the following sequence, or the following sequence A fusion protein having 1-2 amino acid substitutions on the CDRs of an antibody as defined by
(1) the amino acid sequence of the heavy chain variable region of SEQ ID NO: 64; and/or
(2) the amino acid sequence of the light chain variable region of SEQ ID NO: 65.
The fusion protein according to claim 18, wherein the CDRs of the heavy chain variable region and/or the CDRs of the light chain variable region in the Fab of IgG are as follows:
(1) for the heavy chain variable region, the amino acid sequence of CDR1 is selected from amino acid sequences having 1 or 2 amino acid substitutions in SEQ ID NOs: 34-38 and SEQ ID NOs: 34-38; the amino acid sequence of CDR2 is selected from amino acid sequences having 1 or 2 amino acid substitutions in SEQ ID NOs: 39-43 and SEQ ID NOs: 39-43; the amino acid sequence of CDR3 is selected from amino acid sequences having 1 or 2 amino acid substitutions in SEQ ID NOs: 44-48 and SEQ ID NOs: 44-48; and/or
(2) for the light chain variable region, the amino acid sequence of CDR1 is selected from the amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NOs: 49-53 and SEQ ID NOs: 49-53; the amino acid sequence of CDR2 is selected from an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NOs: 54-58 and SEQ ID NOs: 54-58; The amino acid sequence of CDR3 is selected from amino acid sequences having 1 or 2 amino acid substitutions in SEQ ID NOs: 59-63 and SEQ ID NOs: 59-63.
20. The method of claim 19,
(1) the amino acid sequence of CDRs 1-3 of the heavy chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 34, 39, 44 or SEQ ID NO: 34, 39, 44; and/or the amino acid sequence of CDRs 1-3 of the light chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 49, 54, 59, or SEQ ID NO: 49, 54, 59; or
(2) the amino acid sequence of CDRs 1-3 of the heavy chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 35, 40, 45, or SEQ ID NO: 35, 40, 45; and/or the amino acid sequence of CDRs 1-3 of the light chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 50, 55, 60, or SEQ ID NO: 50, 55, 60; or
(3) the amino acid sequence of CDRs 1-3 of the heavy chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 36, 41, 46 or SEQ ID NO: 36, 41, 46; and/or the amino acid sequence of CDRs 1-3 of the light chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 51, 56, 61, or SEQ ID NO: 51, 56, 61; or
(4) the amino acid sequence of CDRs 1-3 of the heavy chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 37, 42, 47, or SEQ ID NO: 37, 42, 47; and/or the amino acid sequence of CDRs 1-3 of the light chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 52, 57, 62, or SEQ ID NO: 52, 57, 62; or
(5) the amino acid sequence of CDRs 1-3 of the heavy chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 38, 43, 48 or SEQ ID NO: 38, 43, 48; and/or the amino acid sequence of CDRs 1-3 of the light chain variable region is an amino acid sequence having 1 or 2 amino acid substitutions in SEQ ID NO: 53, 58, 63, or SEQ ID NO: 53, 58, 63.
The method according to claim 20, wherein the amino acid sequence of CDRs 1-3 of the heavy chain variable region is SEQ ID NO: 36, 41, 46; and / or the amino acid sequence of CDRs 1-3 of the light chain variable region is SEQ ID NO: 51, 56, 61, the fusion protein.
22. The method of claim 21,
(1) the heavy chain variable region has the sequence shown in SEQ ID NO: 64, or CDRs 1-3 identical to SEQ ID NO: 64, and has 80%, 85%, 90%, 95%, 96% homology compared to SEQ ID NO: 64 , 97%, 98%, 99% or more of the sequence; and/or
(2) the light chain variable region has the sequence shown in SEQ ID NO: 65, or CDRs 1-3 identical to SEQ ID NO: 65, and has 80%, 85%, 90%, 95%, 96% homology compared to SEQ ID NO: 65 , 97%, 98%, 99% or more, the fusion protein.
23. The method of claim 22,
(1) the heavy chain of the fusion protein has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence shown in SEQ ID NO: 68, or SEQ ID NO: 68 has a sequence with;
(2) the light chain of the fusion protein has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology compared to the amino acid sequence shown in SEQ ID NO: 69, or SEQ ID NO: 69 A fusion protein that has an abnormal sequence.
24. The method of claim 23,
(1) the amino acid sequence of the heavy chain of the fusion protein has 1-15 amino acid site mutations compared to SEQ ID NO: 68 or has an alternative peptide linker;
(2) The amino acid sequence of the light chain of the fusion protein has 1-10 amino acid site mutations compared to SEQ ID NO: 69, the fusion protein.
23. The method of claim 22,
(1) the heavy chain of the fusion protein has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence shown in SEQ ID NO: 70, or SEQ ID NO: 70 have a sequence with;
(2) the light chain of the fusion protein has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence shown in SEQ ID NO: 71, or SEQ ID NO: 71 A fusion protein having a sequence with
26. The method of claim 25,
(1) the amino acid sequence of the heavy chain of the fusion protein has 1-15 amino acid site mutations compared to SEQ ID NO: 70 or has an alternative peptide linker;
(2) the amino acid sequence of the light chain of the fusion protein has 1-10 amino acid site mutations compared to SEQ ID NO: 71, the fusion protein.
The fusion protein according to any one of claims 1 to 10, wherein the IgG is atezolizumab, avelumab, durvalumab, nivolumab, pembrolizumab, semiplimab or ipilimumab.
28. The fusion protein according to any one of claims 1 to 27, wherein the C-terminus or/and N-terminus of the second binding domain has a peptide linker, wherein the peptide linker consists of 2-30 amino acids. .
29. The method of claim 28, wherein the peptide linker is
(1) (GGGGS)n; or
(2) AKTTPKLEEEGEFSEAR (SEQ ID NO: 80); or
(3) AKTTPKLEEEGEFSEARV (SEQ ID NO: 81); or
(4) AKTTPKLGG (SEQ ID NO: 82); or
(5) SAKTTPKLGG (SEQ ID NO: 83); or
(6) SAKTTP (SEQ ID NO: 84); or
(7) RADAAP (SEQ ID NO: 85); or
(8) RADAAPTVS (SEQ ID NO: 86); or
(9) RADAAAAGGPGS (SEQ ID NO: 87); or
(10) RADAAAA (SEQ ID NO: 88); or
(11) SAKTTPKLEEEGEFSEARV (SEQ ID NO: 89); or
(12) ADAAP (SEQ ID NO: 90); or
(13) DAAPTVSIFPP (SEQ ID NO: 91); or
(14) TVAAP (SEQ ID NO: 92); or
(15) TVAAPSVFIFFP (SEQ ID NO: 93); or
(16) QPKAAP (SEQ ID NO: 94); or
(17) QPKAAPSVTLFPP (SEQ ID NO: 95); or
(18) AKTTPP (SEQ ID NO: 96); or
(19) AKTTPPSVTPLAP (SEQ ID NO: 97); or
(20) AKTTAP (SEQ ID NO: 98); or
(21) AKTTAPSVYPLAP (SEQ ID NO: 99); or
(22) ASTKGP (SEQ ID NO: 100); or
(23) ASTKGPSVFPLAP (SEQ ID NO: 101); or
(24) GENKVEYAPALMALS (SEQ ID NO: 102); or
(25) GPAKELTPLKEAKVS (SEQ ID NO: 103); or
(26) GHEAAAVMQVQYPAS (SEQ ID NO: 104); or
(27) GGGGSGGGGSGGGGSA (SEQ ID NO: 105),
Wherein n is 1, 2, 3 or 4, the fusion protein.
30. The fusion protein according to any one of claims 1 to 29, wherein the size of the inserted second binding domain does not exceed 300 amino acids.
31. The fusion according to any one of claims 1 to 30, wherein the insertion site of the second binding domain is located in the anterior central portion of the hinge region, and the insertion site does not affect disulfide bond formation of the immunoglobulin. protein.
The fusion protein according to claim 31, wherein the front central portion of the hinge region refers to the previous portion of 231A.
The fusion protein according to any one of claims 1 to 32, wherein a part of amino acids in the hinge region before and after the insertion site are substituted or deleted.
34. The fusion protein of claim 33, wherein the hinge region comprises a D221G and/or C220V mutation.
35. The method of any one of claims 1-34, wherein said IgG is selected from mammalian IgG, humanized IgG and human IgG, said mammal including mouse, rat and rabbit; Preferably, the IgG is IgG1, IgG2, IgG3 or IgG4, the fusion protein.
36. The fusion protein according to any one of claims 1 to 35, wherein the Fc region of the fusion protein is aglycosylated or deglycosylated, fucosylation reduced or afucosylated.
37. An isolated polynucleotide encoding a fusion protein according to any one of claims 1 to 36.
A nucleic acid construct comprising the polynucleotide according to claim 37, preferably the nucleic acid construct is a vector.
A host cell comprising the polynucleotide according to claim 37 or the nucleic acid construct or vector according to claim 38, preferably said cell is a prokaryotic cell, eukaryotic cell, yeast cell, mammalian cell, E. coli cell or CHO cell, NS0 cell , an Sp2/0 cell or a BHK cell.
37. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 36 and a pharmaceutically acceptable carrier.
A method of treating a tumor or cancer, comprising administering the fusion protein according to any one of claims 1 to 36 or the pharmaceutical composition according to claim 40 to a subject in need of treatment or alleviation.
The fusion protein according to any one of claims 1 to 36, the polynucleotide according to claim 37, the nucleic acid construct or vector according to claim 38, or the pharmaceutical composition according to claim 40 for the treatment of a tumor or cancer or Use in the manufacture of a medicament for prophylaxis.
43. The use according to claim 42, wherein the tumor or cancer comprises a solid tumor or a non-solid tumor.
A diagnostic kit comprising the fusion protein according to any one of claims 1 to 36.
37. A method for preparing a fusion protein according to any one of claims 1 to 36, comprising:
A method for producing a fusion protein, comprising the steps of culturing the host cell according to claim 39 under conditions allowing expression of the nucleic acid construct according to claim 38, and recovering the produced fusion protein from the culture.

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